Primers and probes for the detection of streptococcus pneumoniae

ABSTRACT

Methods of detecting  Streptococcus pneumoniae  ( S. pneumoniae ), are disclosed. A sample suspected of containing a nucleic acid of  S. pneumoniae  is screened for the presence or absence of that nucleic acid. The presence of the  S. pneumoniae  nucleic acid indicates the presence of  S. pneumoniae . Determining whether the  S. pneumoniae  nucleic acid is present in the sample can be accomplished by detecting hybridization between a  S. pneumoniae  probe, such as a  S. pneumoniae  lytA probe, a  S. pneumoniae  psaA probe, or a  S. pneumoniae  ply probe. Probes and primers for the detection of  S. pneumoniae  are also disclosed. Kits and arrays that contain the disclosed probes and/or primers also are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/938,799 filed May 18, 2007, which is incorporated by reference hereinin its entirety.

FIELD

This disclosure relates to primers and probes for detectingStreptococcus pneumoniae, as well as kits including the probes andprimers and methods of using the probes and primers.

BACKGROUND

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive bacteriaresponsible for considerable morbidity and mortality (particularly inthe young and aged), causing diseases such as pneumonia, bacteremia,meningitis, acute otitis media, and sinusitis. It is estimated the 20%of S. pneumoniae cases lead to bacteremia, and other manifestations suchas meningitis, with a mortality rate close to 30% even with antibiotictreatment. S. pneumoniae is found in the nasopharynx of 11-76% of thepopulation, averaging 40-50% for children and 20-30% for adults (Ghaffaret al., J. Infect. Dis. 18, 638-46, 1999).

Those most commonly at risk for pneumococcal infection are childrenbetween 6 months and 4 years of age and adults over 60 years of age.Virtually every child will experience pneumococcal otitis media beforethe age of 5 years. It is estimated that 25% of all community-acquiredpneumonia is due to pneumococcus (1,000 per 100,000 inhabitants).Recently, epidemics of disease have reappeared in settings such aschronic care facilities, military camps, and day care centers, asituation not recognized since the pre-antibiotic era. S. pneumoniaeremains a significant human pathogen because of the morbidity andmortality it causes in young children, the elderly and inimmunocompromised patients.

The limitations of culture based, conventional S. pneumoniae diagnostictests make definitive diagnosis difficult to establish. For example, theisolation of S. pneumoniae from blood, the recognized definitive testfor the presence of S. pneumoniae may lack sensitive, only givingpositive results in 20-30% of adult cases of pneumococcal pneumonia andless than 10% of children's cases. Serologic assays for both antibodyand antigen detection suffer from a lack of specificity and sensitivity,for example the recently introduced urine antigen test, Binax NOW®,while shown to be sensitive and specific for adults by some studies, isunable to distinguish between carriage and disease in children.

In addition, the misidentification of pneumococcus-like viridansStreptococci (P-LVS) as S. pneumoniae presents additional opportunitiesfor misdiagnosis especially when attempted with non-sterile sitespecimens such as sputum. Identification of S. pneumoniae has typicallybeen based on bile solubility, optochin sensitivity, and GenProbeACCUPROBE® Pneumococcus identification test; but increasingly there havebeen reports of P-LVS isolated from clinical specimens, which may givepositive or variable reactions in one or more of these standardpneumococcal tests. Among a subset of reported isolates of P-LVS, anewly recognized species, classified as S. pseudopneumoniae (Spseudo),has been described and characterized (Arbique et al., J. Clin.Microbiol. 42: 4686-4696, 2004). Spseudo organisms are bile solubilitynegative and resistant to optochin in the presence of 5% CO₂, but areACCUPROBE® positive (Arbique et al., J. Clin. Microbiol. 42: 4686-4696,2004) and thus yield a false positive for S. pneumoniae infection.

The appearance of these pneumococcus-like organisms has complicatedidentification and diagnosis even further, especially when non-sterilesite respiratory specimens are used for making determinations.Therefore, special care must be taken to monitor and correctly identifyconfirmed pneumococci in the clinical setting. Thus, to make an accuratediagnosis the need exists for assays that can discriminate between S.pneumoniae and the Spseudo and other P-LVS species. The presentdisclosure meets this need by providing assays that can discriminatebetween S. pneumoniae and other organisms while still retaining highsensitivity for S. pneumoniae.

SUMMARY

Accurate diagnosis of pneumococcal disease is frequently hampered by themisidentification of pneumococcus-like viridans streptococci species(P-LVS) as Streptococcus pneumoniae (S. pneumoniae). In order to achievean accurate diagnosis, an assay should be both sensitive and highlyspecific for diagnosis of the disease, such as the diagnosis of a S.pneumoniae infection. By analyzing regions of the lytA, ply, and psaAgenes that are unique S. pneumoniae, assays disclosed herein have beendeveloped that are highly specific for S. pneumoniae while retaininghigh sensitivity for S. pneumoniae. Because the disclosed assays areboth sensitive and specific, the presence of S. pneumoniae innon-sterile samples, such as samples obtained from a subject, can bereliably determined. Such accuracy is important for medicalprofessionals to make the best possible diagnosis and treatment plan,particularly in the age of over-prescription of medication, such asantibiotics. In addition, because the assays are highly specific fornucleic acids from S. pneumoniae and do not show cross reaction with thenucleic acids from other organisms the assay is well suited tomultiplexing, for example in a multiplex real-time PCR assay fordetecting multiple pathogens that may be present in a sample, such as asample obtained from a subject.

The present disclosure relates to methods of detecting the presence ofStreptococcus pneumoniae (S. pneumoniae) nucleic acids in a sample, suchas a biological sample obtained from a subject, for example to detect S.pneumoniae in the sample. The disclosed methods can be used fordiagnosing an S. pneumoniae infection, for example in a subjectsuspected of having an S. pneumoniae infection, by analyzing abiological specimen from a subject to detect a broad variety of S.pneumoniae nucleic acids, such as S. pneumoniae lytA, ply, and psaAnucleic acids using the probes and/or primers disclosed herein. Inaddition, the probes and primers provided permit the rapid evaluation ofa subject with an apparent S. pneumoniae infection by quicklydetermining whether the infection is caused by S. pneumoniae or anotherorganism. This rapid evaluation involves ruling out the presence of S.pneumoniae, ruling in the presence of S. pneumoniae, or a combination ofboth, for example in a multiplex real-time PCR assay.

In some embodiments, the method involves hybridizing an S. pneumoniaenucleic acid to an S. pneumoniae specific probe between 20 and 40nucleotides in length, and detecting hybridization between the S.pneumoniae nucleic acid and the probe. In some embodiments, the probe isdetectably labeled. In some embodiments, the probe is capable ofhybridizing under conditions of very high stringency to a S. pneumoniaenucleic acid sequence set forth as SEQ ID NO: 13 (the lytA gene from S.pneumoniae), SEQ ID NO: 14 (the psaA gene from S. pneumoniae), or SEQ IDNO: 15 (the ply gene from S. pneumoniae). In specific embodiments, theprobe includes a nucleic acid sequence that is at least 95% identical toa nucleic acid sequence set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 9, or SEQ ID NO: 12.

The present disclosure also relates to methods of detecting and/ordiscriminating between S. pneumoniae or another organism, such as abacterial organism, for example pneumococcus-like viridans Streptococci(P-LVS), such as S. pseudopneumoniae (Spseudo).

In some embodiments, the methods disclosed herein include amplifying theS. pneumoniae nucleic acids with at least one primer specific for a S.pneumoniae nucleic acid. In some embodiments, the primer specific for aS. pneumoniae nucleic acid is 15 to 40 nucleotides in length and iscapable of hybridizing under very high stringency conditions to a S.pneumoniae nucleic acid sequence set forth as SEQ ID NO: 13, SEQ ID NO:14, or SEQ ID NO: 15. In some embodiments, the primer specific for a S.pneumoniae nucleic acid is 15 to 40 nucleotides in length and includes anucleic acid sequence at least 95% identical to the nucleotide sequenceset forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 10, or SEQ ID NO: 11.

In some embodiments, the S. pneumoniae nucleic acid is amplified usingat least one primer, such as a pair of primers, specific for a S.pneumoniae gene, such as a S. pneumoniae lytA, psaA, or ply gene. Insome examples, a primer specific for S. pneumoniae lytA includes anucleic acid sequence at least 95% identical to the nucleic acidsequence set forth as one of SEQ ID NO: 3 or SEQ ID NO: 4. In otherexamples, a primer specific for S. pneumoniae psaA includes a nucleicacid sequence at least 95% identical to the nucleic acid sequence setforth as one of SEQ ID NO: 7 or SEQ ID NO: 8. In other examples, aprimer specific for S. pneumoniae ply includes a nucleic acid sequenceat least 95% identical to the nucleic acid sequence set forth as one ofSEQ ID NO: 10 or SEQ ID NO: 11.

This disclosure also relates to probes capable of hybridizing to S.pneumoniae nucleic acids, such as S. pneumoniae lytA, psaA, or plynucleic acids. In some embodiments, these probes are between 20 and 40nucleotides in length and capable of hybridizing under very highstringency conditions to a S. pneumoniae nucleic acid sequence set forthas SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. In several examples,these probes are between 20 and 40 nucleotides in length and include anucleic acid sequence set forth as SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 9, or SEQ ID NO: 12.

This disclosure also relates to primers capable of hybridizing to andamplifying S. pneumoniae nucleic acids, such as S. pneumoniae lytA,psaA, or ply nucleic acids. In some embodiments, these primers arebetween 20 and 40 nucleotides in length and capable of hybridizing undervery high stringency conditions to a S. pneumoniae nucleic acid sequenceset forth as SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15. In severalexamples, these primers are 15 to 40 nucleotides in length and include anucleic acid sequence at least 95% identical to a nucleic acid sequenceset forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 11, or SEQ ID NO: 12.

The disclosure also provides devices, such as arrays, as well as kitsfor detecting S. pneumoniae nucleic acids in a sample suspected ofcontaining S. pneumoniae.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a generalized procedure forhybridizing a S. pneumoniae specific probe to a S. pneumoniae nucleicacid.

FIG. 2 is a schematic representation of a generalized procedure forhybridizing a S. pneumoniae specific probe to a S. pneumoniae nucleicacid, wherein the S. pneumoniae nucleic acid is initially a doublestranded nucleic acid.

FIG. 3 is a schematic representation of a generalized procedure forhybridizing and detecting S. pneumoniae using a S. pneumoniae specificTAQMAN® probe.

FIG. 4 is a graph of theoretical data generated from real-timepolymerase chain reaction (real-time PCR) using TAQMAN® probes.

FIG. 5 is a graph of S. pneumoniae specific TAQMAN® probes used in amultiplex real-time PCR assay, showing the efficiency of the indicatedprobe and primer sets.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and one letter code for amino acids, as defined in 37 C.F.R.§1.822. If only one strand of each nucleic acid sequence is shown, thecomplementary strand is understood as included by any reference to thedisplayed strand.

SEQ ID NO: 1 is the nucleotide sequence of a theoretical oligo.

SEQ ID NO: 2 is the nucleotide sequence of a theoretical oligo.

SEQ ID NO: 3 is the nucleotide sequence of a Streptococcus pneumoniaelytA forward real-time PCR primer.

SEQ ID NO: 4 is the nucleotide sequence of a Streptococcus pneumoniaelytA reverse real-time PCR primer.

SEQ ID NO: 5 is the nucleotide sequence of a Streptococcus pneumoniaelytA real-time PCR probe.

SEQ ID NO: 6 is the nucleotide sequence of a Streptococcus pneumoniaelytA real-time PCR probe.

SEQ ID NO: 7 is the nucleotide sequence of a Streptococcus pneumoniaepsaA forward real-time PCR primer.

SEQ ID NO: 8 is the nucleotide sequence of a Streptococcus pneumoniaepsaA reverse real-time PCR primer.

SEQ ID NO: 9 is the nucleotide sequence of a Streptococcus pneumoniaepsaA real-time PCR probe.

SEQ ID NO: 10 is the nucleotide sequence of a Streptococcus pneumoniaeply forward real-time PCR primer.

SEQ ID NO: 11 is the nucleotide sequence of a Streptococcus pneumoniaeply reverse real-time PCR primer.

SEQ ID NO: 12 is the nucleotide sequence of a Streptococcus pneumoniaeply real-time PCR probe.

SEQ ID NO: 13 is an exemplary nucleotide sequence of Streptococcuspneumoniae lytA.

SEQ ID NO: 14 is an exemplary nucleotide sequence of Streptococcuspneumoniae psaA.

SEQ ID NO: 15 is an exemplary nucleotide sequence of Streptococcuspneumoniae ply.

DETAILED DESCRIPTION I. Explanation of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “a probe” includes single or pluralprobes and can be considered equivalent to the phrase “at least oneprobe.”

As used herein, the term “comprises” means “includes.” Thus, “comprisinga probe” means “including a probe” without excluding other elements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the invention, thefollowing explanations of terms are provided:

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and veterinary subjects.

Amplification: To increase the number of copies of a nucleic acidmolecule. The resulting amplification products are called “amplicons.”Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample, for example the number of copies of aS. pneumoniae nucleic acid, such as a S. pneumoniae lytA nucleic acid orfragment thereof. An example of amplification is the polymerase chainreaction (PCR), in which a sample is contacted with a pair ofoligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Other examples of in vitro amplification techniques include quantitativereal-time PCR; reverse transcriptase PCR (RT-PCR); real-time PCR;real-time reverse transcriptase PCR (rt RT-PCR); nested PCR; stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881, repair chain reaction amplification (see WO 90/01069); ligasechain reaction amplification (see European patent publication EP-A-320308); gap filling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription-free amplification (see U.S.Pat. No. 6,025,134) amongst others.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA alsocan contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA can besynthesized in the laboratory by reverse transcription from RNA, forexample an RNA from S. pneumoniae, such as an RNA encoding S. pneumoniaelytA, psaA, or ply.

Change: To become different in some way, for example to be altered, suchas increased or decreased. A detectable change is one that can bedetected, such as a change in the intensity, frequency or presence of anelectromagnetic signal, such as fluorescence, for example a change influorescence of a probe, such as an TAQMAN® probe specific for an S.pneumoniae nucleic acid, such as a S. pneumoniae lytA nucleic acid, a S.pneumoniae psaA nucleic acid, or a S. pneumoniae ply nucleic acid. Insome examples, the detectable change is a reduction in fluorescenceintensity. In some examples, the detectable change is an increase influorescence intensity.

Complementary: A double-stranded DNA or RNA strand consists of twocomplementary strands of base pairs. Complementary binding occurs whenthe base of one nucleic acid molecule forms a hydrogen bond to the baseof another nucleic acid molecule. Normally, the base adenine (A) iscomplementary to thymidine (T) and uracil (U), while cytosine (C) iscomplementary to guanine (G). For example, the sequence 5′-ATCG-3′ ofone ssDNA molecule can bond to 3′-TAGC-5′ of another ssDNA to form adsDNA. In this example, the sequence 5′-ATCG-3′ is the reversecomplement of 3′-TAGC-5′.

Nucleic acid molecules can be complementary to each other even withoutcomplete hydrogen-bonding of all bases of each molecule. For example,hybridization with a complementary nucleic acid sequence can occur underconditions of differing stringency in which a complement will bind atsome but not all nucleotide positions. In some examples, a nucleic acidmolecule, such as the probes and primers specific for S. pneumoniaelytA, psaA, or ply disclosed herein, are complementary to a S.pneumoniae lytA, psaA, or ply nucleic acid molecule or the amplificationproducts of such a nucleic acid molecule.

Detect: To determine if an agent (such as a signal, particularnucleotide, amino acid, nucleic acid molecule, and/or organism) ispresent or absent, for example S. pneumoniae. In some examples, this canfurther include quantification. For example, use of the disclosed probesin particular examples permits detection of a fluorophore, for example,detection of a signal from a fluorophore, which can be used to determineif a nucleic acid corresponding to nucleic acid of S. pneumoniae (suchas a S. pneumoniae lytA nucleic acid molecule, a S. pneumoniae psaAnucleic acid molecule, or a S. pneumoniae ply nucleic acid molecule) ispresent. The detection of a S. pneumoniae nucleic acid moleculeindicates the presence of S. pneumoniae in the sample, for example a S.pneumoniae infection in the sample.

Electromagnetic radiation: A series of electromagnetic waves that arepropagated by simultaneous periodic variations of electric and magneticfield intensity, and that includes radio waves, infrared, visible light,ultraviolet light, X-rays and gamma rays. In particular examples,electromagnetic radiation is emitted by a laser, which can possessproperties of monochromaticity, directionality, coherence, polarization,and intensity. Lasers are capable of emitting light at a particularwavelength (or across a relatively narrow range of wavelengths), forexample, so that energy from the laser can excite a donor but not anacceptor fluorophore.

Emission or emission signal: The light of a particular wavelengthgenerated from a source. In particular examples, an emission signal isemitted from a fluorophore after the fluorophore absorbs light at itsexcitation wavelength(s).

Excitation or excitation signal: The light of a particular wavelengthnecessary and/or sufficient to excite an electron transition to a higherenergy level. In particular examples, an excitation is the light of aparticular wavelength necessary and/or sufficient to excite afluorophore to a state such that the fluorophore will emit a different(such as a longer) wavelength of light then the wavelength of light fromthe excitation signal.

Fluorophore: A chemical compound, which when excited by exposure to aparticular stimulus such as a defined wavelength of light, emits light(fluoresces), for example at a different wavelength (such as a longerwavelength of light).

Fluorophores are part of the larger class of luminescent compounds.Luminescent compounds include chemiluminescent molecules, which do notrequire a particular wavelength of light to luminesce, but rather use achemical source of energy. Therefore, the use of chemiluminescentmolecules (such as aequorin) eliminates the need for an external sourceof electromagnetic radiation, such as a laser.

Examples of particular fluorophores that can be used in the S.pneumoniae specific probes and primers disclosed herein are known tothose of skill in the art and include those provided in U.S. Pat. No.5,866,366 to Nazarenko et al., such as4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid; acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide; BrilliantYellow; coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), QFITC (XRITC), -6-carboxy-fluorescein(HEX), and TET (Tetramethyl fluorescein); fluorescamine; IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (CIBACRON™. Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate,N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,and tetramethyl rhodamine isothiocyanate (TRITC); sulforhodamine B;sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine101 (Texas Red); riboflavin; rosolic acid and terbium chelatederivatives; LightCycler Red 640; Cy5.5; and Cy56-carboxyfluorescein;boron dipyrromethene difluoride (BODIPY); acridine; stilbene;6-carboxy-X-rhodamine (ROX); Texas Red; Cy3; Cy5, VIC® (AppliedBiosystems); LC Red 640; LC Red 705; and Yakima yellow amongst others.

Other suitable fluorophores include those known to those skilled in theart, for example those available from Molecular Probes (Eugene, Oreg.).In particular examples, a fluorophore is used as a donor fluorophore oras an acceptor fluorophore.

“Acceptor fluorophores” are fluorophores which absorb energy from adonor fluorophore, for example in the range of about 400 to 900 nm (suchas in the range of about 500 to 800 nm). Acceptor fluorophores generallyabsorb light at a wavelength which is usually at least 10 nm higher(such as at least 20 nm higher) than the maximum absorbance wavelengthof the donor fluorophore, and have a fluorescence emission maximum at awavelength ranging from about 400 to 900 nm. Acceptor fluorophores havean excitation spectrum that overlaps with the emission of the donorfluorophore, such that energy emitted by the donor can excite theacceptor. Ideally, an acceptor fluorophore is capable of being attachedto a nucleic acid molecule.

In a particular example, an acceptor fluorophore is a dark quencher,such as Dabcyl, QSY7 (Molecular Probes), QSY33 (Molecular Probes), BLACKHOLE QUENCHERS™ (Glen Research), ECLIPSE™ Dark Quencher (EpochBiosciences), or IOWA BLACK™ (Integrated DNA Technologies). A quenchercan reduce or quench the emission of a donor fluorophore. In such anexample, instead of detecting an increase in emission signal from theacceptor fluorophore when in sufficient proximity to the donorfluorophore (or detecting a decrease in emission signal from theacceptor fluorophore when a significant distance from the donorfluorophore), an increase in the emission signal from the donorfluorophore can be detected when the quencher is a significant distancefrom the donor fluorophore (or a decrease in emission signal from thedonor fluorophore when in sufficient proximity to the quencher acceptorfluorophore).

“Donor Fluorophores” are fluorophores or luminescent molecules capableof transferring energy to an acceptor fluorophore, thereby generating adetectable fluorescent signal from the acceptor. Donor fluorophores aregenerally compounds that absorb in the range of about 300 to 900 nm, forexample about 350 to 800 nm. Donor fluorophores have a strong molarabsorbance coefficient at the desired excitation wavelength, for examplegreater than about 10³ M⁻¹ cm⁻¹.

Fluorescence Resonance Energy Transfer (FRET): A spectroscopic processby which energy is passed between an initially excited donor to anacceptor molecule separated by 10-100 Å. The donor molecules typicallyemit at shorter wavelengths that overlap with the absorption of theacceptor molecule. The efficiency of energy transfer is proportional tothe inverse sixth power of the distance (R) between the donor andacceptor (1/R⁶) fluorophores and occurs without emission of a photon. Inapplications using FRET, the donor and acceptor dyes are different, inwhich case FRET can be detected either by the appearance of sensitizedfluorescence of the acceptor or by quenching of donor fluorescence. Forexample, if the donor's fluorescence is quenched it indicates the donorand acceptor molecules are within the Förster radius (the distance whereFRET has 50% efficiency, about 20-60 Å), whereas if the donor fluorescesat its characteristic wavelength, it denotes that the distance betweenthe donor and acceptor molecules has increased beyond the Försterradius, such as when a TAQMAN® probe is degraded by Taq polymerasefollowing hybridization of the probe to a target nucleic acid sequenceor when a hairpin probe is hybridized to a target nucleic acid sequence.In another example, energy is transferred via FRET between two differentfluorophores such that the acceptor molecule can emit light at itscharacteristic wavelength, which is always longer than the emissionwavelength of the donor molecule.

Examples of oligonucleotides using FRET that can be used to detectamplicons include linear oligoprobes, such as HybProbes, 5′ nucleaseoligoprobes, such as TAQMAN® probes, hairpin oligoprobes, such asmolecular beacons, scorpion primers and UniPrimers, minor groove bindingprobes, and self-fluorescing amplicons, such as sunrise primers.

Hybridization: The ability of complementary single-stranded DNA or RNAto form a duplex molecule (also referred to as a hybridization complex).Nucleic acid hybridization techniques can be used to form hybridizationcomplexes between a probe or primer and a nucleic acid, such as a S.pneumoniae nucleic acid molecule, such as a S. pneumoniae lytA, a psaA,or a ply nucleic acid molecule. For example, a probe or primer (such asany of SEQ ID NOs:3-12) having some homology to a S. pneumoniae nucleicacid molecule will form a hybridization complex with a S. pneumoniaenucleic acid molecule (such as any of SEQ ID NOs:13-15).

With reference to FIG. 1, the formation of hybridization complex 110occurs between single stranded probe 105 and single stranded targetnucleic acid 100 (such as a S. pneumoniae nucleic acid molecule, forexample a lytA, psaA, or ply nucleic acid molecule). With reference toFIG. 2, when target nucleic acid 210 is initially one strand of duplexnucleic acid 200 the duplex must be melted (at least partially) intotarget nucleic acid 210 and complementary strand 220 for probe 205 tohybridize and form hybridization complex 230.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength (suchas the Na+ concentration) of the hybridization buffer will determine thestringency of hybridization. Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning, second edition, ColdSpring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). Thefollowing is an exemplary set of hybridization conditions and is notlimiting:

Very High Stringency (Detects Sequences that Share at Least 90%Identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share at Least 80% Identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share at Least 50% Identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

The probes and primers disclosed herein can hybridize to S. pneumoniaenucleic acid molecules, such as a lytA, psaA, or ply nucleic acidmolecule, under low stringency, high stringency, and very highstringency conditions.

Isolated: An “isolated” biological component (such as a nucleic acid)has been substantially separated or purified away from other biologicalcomponents in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA, RNA, and proteins. Nucleic acidsthat have been “isolated” include nucleic acids purified by standardpurification methods. The term also embraces nucleic acids prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids, such as probes and primers, for example S. pneumoniaespecific probes and primers disclosed herein. Isolated does not requireabsolute purity, and can include nucleic acid molecules that are atleast 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, oreven 100% isolated.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy. For example, a label can be attached to anucleotide, thereby permitting detection of the nucleotide, such asdetection of the nucleic acid molecule of which the nucleotide is apart, such as a S. pneumoniae specific probe and/or primer. Examples oflabels include, but are not limited to, radioactive isotopes, enzymesubstrates, co-factors, ligands, chemiluminescent agents, fluorophores,haptens, enzymes, and combinations thereof. Methods for labeling andguidance in the choice of labels appropriate for various purposes arediscussed for example in Sambrook et al. (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

Nucleic acid (molecule or sequence): A deoxyribonucleotide orribonucleotide polymer including without limitation, cDNA, mRNA, genomicDNA, and synthetic (such as chemically synthesized) DNA or RNA. Thenucleic acid can be double stranded (ds) or single stranded (ss). Wheresingle stranded, the nucleic acid can be the sense strand or theantisense strand. Nucleic acids can include natural nucleotides (such asA, T/U, C, and G), and can include analogs of natural nucleotides, suchas labeled nucleotides. In some examples, a nucleic acid is a S.pneumoniae nucleic acid, which can include nucleic acids purified fromS. pneumoniae as well as the amplification products of such nucleicacids.

Nucleotide: The fundamental unit of nucleic acid molecules. A nucleotideincludes a nitrogen-containing base attached to a pentose monosaccharidewith one, two, or three phosphate groups attached by ester linkages tothe saccharide moiety.

The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP orA), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U).

Nucleotides include those nucleotides containing modified bases,modified sugar moieties and modified phosphate backbones, for example asdescribed in U.S. Pat. No. 5,866,336 to Nazarenko et al. (hereinincorporated by reference).

Examples of modified base moieties which can be used to modifynucleotides at any position on its structure include, but are notlimited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N˜6-sopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine amongstothers.

Examples of modified sugar moieties, which may be used to modifynucleotides at any position on its structure, include, but are notlimited to arabinose, 2-fluoroarabinose, xylose, and hexose, or amodified component of the phosphate backbone, such as phosphorothioate,a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, or aformacetal or analog thereof.

Primers: Short nucleic acid molecules, such as a DNA oligonucleotide,for example sequences of at least 15 nucleotides, which can be annealedto a complementary target nucleic acid molecule by nucleic acidhybridization to form a hybrid between the primer and the target nucleicacid strand. A primer can be extended along the target nucleic acidmolecule by a polymerase enzyme. Therefore, primers can be used toamplify a target nucleic acid molecule (such as a portion of a S.pneumoniae nucleic acid molecule, for example a portion of a lytA, psaA,or ply nucleic acid molecule), wherein the sequence of the primer isspecific for the target nucleic acid molecule, for example so that theprimer will hybridize to the target nucleic acid molecule under veryhigh stringency hybridization conditions.

The specificity of a primer increases with its length. Thus, forexample, a primer that includes 30 consecutive nucleotides will annealto a target sequence with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, to obtain greater specificity,probes and primers can be selected that include at least 15, 20, 25, 30,35, 40, 45, 50 or more consecutive nucleotides.

In particular examples, a primer is at least 15 nucleotides in length,such as at least 15 contiguous nucleotides complementary to a targetnucleic acid molecule. Particular lengths of primers that can be used topractice the methods of the present disclosure (for example, to amplifya region of a S. pneumoniae nucleic acid molecule, such as a portion ofa lytA, psaA, or ply nucleic acid molecule) include primers having atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 45, at least50, or more contiguous nucleotides complementary to the target nucleicacid molecule to be amplified, such as a primer of 15-60 nucleotides,15-50 nucleotides, 20-40 nucleotides, 25-50, nucleotides, or 15-30nucleotides.

Primer pairs can be used for amplification of a nucleic acid sequence,for example, by PCR, real-time PCR, or other nucleic-acid amplificationmethods known in the art. An “upstream” or “forward” primer is a primer5′ to a reference point on a nucleic acid sequence. A “downstream” or“reverse” primer is a primer 3′ to a reference point on a nucleic acidsequence. In general, at least one forward and one reverse primer areincluded in an amplification reaction. PCR primer pairs can be derivedfrom a known sequence (such as the S. pneumoniae nucleic acid sequencesset forth as SEQ ID NOS:13-15), for example, by using computer programsintended for that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.) or PRIMER EXPRESS®Software (Applied Biosystems, AB, Foster City, Calif.).

Methods for preparing and using primers are described in, for example,Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y.; Ausubel et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences. In oneexample, a primer includes a label.

Probe: A probe comprises an isolated nucleic acid capable of hybridizingto a target nucleic acid (such as a S. pneumoniae nucleic acid, forexample a S. pneumoniae lytA, psaA, or ply nucleic acid molecule). Adetectable label or reporter molecule can be attached to a probe.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes.

Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley-Intersciences (1987).

In a particular example, a probe includes at least one fluorophore, suchas an acceptor fluorophore or donor fluorophore. For example, afluorophore can be attached at the 5′- or 3′-end of the probe. Inspecific examples, the fluorophore is attached to the base at the 5′-endof the probe, the base at its 3′-end, the phosphate group at its 5′-endor a modified base, such as a T internal to the probe.

Probes are generally at least 20 nucleotides in length, such as at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 41, at least42, at least 43, at least 44, at least 45, at least 46, at least 47, atleast 48, at least 49, at least 50 at least 51, at least 52, at least53, at least 54, at least 55, at least 56, at least 57, at least 58, atleast 59, at least 60, or more contiguous nucleotides complementary tothe target nucleic acid molecule, such as 20-60 nucleotides, 30-60nucleotides, 20-50 nucleotides, 30-50 nucleotides, 20-40 nucleotides, or20-30 nucleotides.

Polymerizing agent: A compound capable of reacting monomer molecules(such as nucleotides) together in a chemical reaction to form linearchains or a three-dimensional network of polymer chains. A particularexample of a polymerizing agent is polymerase, an enzyme which catalyzesthe 5′ to 3′ elongation of a primer strand complementary to a nucleicacid template. Examples of polymerases that can be used to amplify anucleic acid molecule include, but are not limited to the E. coli DNApolymerase I, specifically the Klenow fragment which has 3′ to 5′exonuclease activity, Taq polymerase, reverse transcriptase (such asHIV-1 RT), E. coli RNA polymerase, and wheat germ RNA polymerase II.

The choice of polymerase is dependent on the nucleic acid to beamplified. If the template is a single-stranded DNA molecule, aDNA-directed DNA or RNA polymerase can be used; if the template is asingle-stranded RNA molecule, then a reverse transcriptase (such as anRNA-directed DNA polymerase) can be used.

Quantitating a nucleic acid molecule: Determining or measuring aquantity (such as a relative quantity) of nucleic acid moleculespresent, such as the number of amplicons or the number of nucleic acidmolecules present in a sample. In particular examples, it is determiningthe relative amount or actual number of nucleic acid molecules presentin a sample, such as S. pneumoniae nucleic acid molecules present in asample.

Quenching of fluorescence: A reduction of fluorescence. For example,quenching of a fluorophore's fluorescence occurs when a quenchermolecule (such as the fluorescence quenchers listed above) is present insufficient proximity to the fluorophore that it reduces the fluorescencesignal (for example, prior to the binding of a probe to an S. pneumoniaenucleic acid sequence, when the probe contains a fluorophore and aquencher).

Real-time PCR: A method for detecting and measuring products generatedduring each cycle of a PCR, which are proportionate to the amount oftemplate nucleic acid prior to the start of PCR. The informationobtained, such as an amplification curve, can be used to determine thepresence of a target nucleic acid (such as a S. pneumoniae nucleic acid)and/or quantitate the initial amounts of a target nucleic acid sequence.Exemplary procedures for real-time PCR can be found in “Quantitation ofDNA/RNA Using Real-Time PCR Detection” published by Perkin Elmer AppliedBiosystems (1999) and to PCR Protocols (Academic Press New York, 1989).

In some examples, the amount of amplified target nucleic acid (such as aS. pneumoniae nucleic acid molecule for example a S. pneumoniae lytA,psaA, or ply nucleic acid molecule) is detected using a labeled probe,such as a probe labeled with a fluorophore, for example a TAQMAN® probe.In this example, the increase in fluorescence emission is measured inreal-time, during the course of the real-time PCR. This increase influorescence emission is directly related to the increase in targetnucleic acid amplification (such as S. pneumoniae nucleic acidamplification). In some examples, the change in fluorescence (dRn) iscalculated using the equation dRn=Rn⁺−Rn⁻, with Rn⁺ being thefluorescence emission of the product at each time point and Rn⁻ beingthe fluorescence emission of the baseline. The dRn values are plottedagainst cycle number, resulting in amplification plots for each sampleas illustrated in FIG. 4. With reference to FIG. 4, the threshold value(CO is the PCR cycle number at which the fluorescence emission (dRn)exceeds a chosen threshold, which is typically 10 times the standarddeviation of the baseline (this threshold level can, however, be changedif desired).

The threshold cycle is when the system begins to detect the increase inthe signal associated with an exponential growth of PCR product duringthe log-linear phase. This phase provides information about thereaction. The slope of the log-linear phase is a reflection of theamplification efficiency. The efficiency of the reaction can becalculated by the following equation: E=10^((−1/slope))−1. Theefficiency of the PCR should be 90-100% meaning doubling of the ampliconat each cycle. This corresponds to a slope of −3.1 to −3.6 in the C_(t)vs. log-template amount standard curve. In order to obtain accurate andreproducible results, reactions should have efficiency as close to 100%as possible (meaning a two-fold increase of amplicon at each cycle).

Sample: A sample, such as a biological sample, is a sample obtained froma plant or animal subject. As used herein, biological samples includeall clinical samples useful for detection S. pneumoniae infection insubjects, including, but not limited to, cells, tissues, and bodilyfluids, such as: blood; derivatives and fractions of blood, such asserum; extracted galls; biopsied or surgically removed tissue, includingtissues that are, for example, unfixed, frozen, fixed in formalin and/orembedded in paraffin; tears; milk; skin scrapes; surface washings;urine; sputum; cerebrospinal fluid; prostate fluid; pus; bone marrowaspirates; middle ear fluids, bronchoalveolar levage, trachealaspirates, sputum, nasopharyngeal aspirates, oropharyngeal aspirates, orsaliva. In particular embodiments, the biological sample is obtainedfrom an animal subject, such as in the form of middle ear fluids,bronchoalveolar levage, tracheal aspirates, sputum, nasopharyngealaspirates, oropharyngeal aspirates, or saliva.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn, and tblastx. Blastn is used tocompare nucleic acid sequences, while blastp is used to compare aminoacid sequences. Additional information can be found at the NCBI website.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1554 nucleotidesis 75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(i.e., 15÷20*100=75).

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions. Stringent conditions are sequence-dependent and aredifferent under different environmental parameters.

The nucleic acid probes and primers disclosed herein are not limited tothe exact sequences shown, as those skilled in the art will appreciatethat changes can be made to a sequence, and not substantially affect theability of the probe or primer to function as desired. For example,sequences having at least 80%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, or at least 99%, such as 100% sequenceidentity to any of SEQ ID NOs:3-12 are provided herein. One of skill inthe art will appreciate that these sequence identity ranges are providedfor guidance only; it is possible that probes and primer can be usedthat fall outside these ranges.

Signal: A detectable change or impulse in a physical property thatprovides information. In the context of the disclosed methods, examplesinclude electromagnetic signals such as light, for example light of aparticular quantity or wavelength. In certain examples, the signal isthe disappearance of a physical event, such as quenching of light.

Streptococcus pneumoniae N-acetylmuramoyl-L-alanine amidase (lytA): ThelytA-encoded autolysin of S. pneumoniae. As used herein “lytA” refers tothe nucleotide sequence of lytA, thus a probe or primer for lytA, suchas those disclosed herein, is capable of hybridizing to the nucleotidesequence of lytA, such as the lytA nucleotide sequence given below (orthe complement thereof). An exemplary nucleotide sequence of lytA asfound at GENBANK® Accession number AE005672 on May 7, 2007 is shownbelow:

(SEQ ID NO: 13) ttattttactgtaatcaagccatctggctctactgtgaattctggcttgtctgccagtgttccgtctggtttgaggtagtaccagcctgttccgtccgctgactggataaaggcatttgataccatggcgccttctttagcgtctaagtagtaccaagtgtccttgtacttgacccagcctgtcttcatggcaccttcttcgttgaaatagtaccacttatcagcgattttcttccagcctgtagccatttcgcctgagttgtcgaaccagtaccagttgccgtctgtgtgcttcctccagcggtctgcaagcatatagcctgaactgtcaaagtagtaccaagtgccattgattttctcaaacttgtcttttggataagagccgtctgaatgtacgtaccagtagccagtgtcattcttctgccagcctgtttcaatcgtcaagccgttctcaatatcatgcttaaactgctcacggctaatgccccatttagcaagatatggataagggtcaacgtggtctgagtggttgtttggttggttattcgtgcaatactcgtgcgttttaattccagctaaactccctgtatcaagcgttttcggcaaacctgcttcatctgctagattgcgtaagagttcgatataaaggcggtagtccgtcatgaactcttctttggttgaatggctttcaatcagttcaaccgctgcataggtctcagcattccaaccgcccccaacgtcccaggcaccattatcaacaggtcctacctgcatgatgcaaccgttcccaacaatgtgcgagaaaaaacctaattctgggtctttccgccagtgataatccgcttcattctgtacggttgaatgcggattcccagttgagtgtgcgtgtacttgcctatatggttgcacgccgacttgaggcaaatctgttcttaatttactcacattaa tttccat

Streptococcus pneumoniae pneumolysin (ply): A virulence factor of S.pneumoniae, is one of the members of thiol-activated cytolysins (TACYs)consisting of four domains. As used herein “ply” refers to thenucleotide sequence of ply, thus a probe or primer for ply, such asthose disclosed herein, is capable of hybridizing to the nucleotidesequence of ply, such as the ply nucleotide sequence given below (or thecomplement thereof). An exemplary nucleotide sequence of ply as found atGENBANK® Accession number AE008539 on May 7, 2007 is shown below:

(SEQ ID NO: 14) ctagtcattttctaccttatcctctacctgaggatagagagttgttccccaaatagaaatcgtccgcttacgcactagtggcaaatcggttttttcataaaccgtacgccaccattcccaggcaagcccggtacactctctaattttgacagagagattacgaacattcccttttaaaggaatactagtggtaaagtgagccgtcaaatcctgcccatttctgtcccaagccttaggagtcaagacttccttaccttgatgatcataggataattcatcccaagtaatataatattgggcaacataggcaccactatgatccagcagtaaatctccgtttctgtaagctgtaaccttagtctcaacatagtctgtactgttttgaaaggtcgcaactacattgtcacgtaaaaaagaagttgtataggaaatcggcaagcctggatgatctgctgtaaagcgactgccttcttgaatcaagtcctctaccatatccaccttgcctgttacaactcgggcacccgaacttgggtcgccccctaaaataaccgccttcacttctgtattgtccaaaatctgcttccactctgtctgaggagctaccttgactccttttatcaaagcttcaaaagcagcctctacttcatcactcttactcgtggtttccaacttgagatagacttggcgcccataagcaacactcgaaatatagaccaaaggacgctctgcagaaattcctctctgttttaaatcctctaccgttacagtatcttgaaacacatctcctggatttttaacagcgtctacgctgactgtataataaatctgcttaaaattaacaatctgaatctgcttttcacctgaatggacagagttaaaatcaatatcaagagaattccctgtcttttcaaagtcagaaccaaacttgaccttgagttgttccatgctgtgagccgttattttttcatactgcattctagctgggacattattgacctgaccataatcttgatgccacttagccaacaaatcgtttaccgctccgcgaacacttgaattgctggggtcttccacttggagaaagctatcgctacttgccaaaccaggcaaatcaatactataagtcatcggagcacgatcaaccgcaagaagagtgggattattctctaacaaggtctcatccactacgagaagtgctccaggatagaggcgactgtcgttggtagctgttacagaaatatcacttgtatttgtcgacaagctccgcttctttctttcgataacaacaaactcatcgggtagctgattaccctctttgatgaaacgattttcaatactttctccctgatgggtcaagagtttctttttatcgtaattcatagctagtataaagtcattta ctgctttatttgccat

Streptococcus pneumoniae surface adhesin A precursor (psaA): psaAencodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II)transport complex. As used herein “psaA” refers to the nucleotidesequence of psaA, thus a probe or primer for psaA, such as thosedisclosed herein, is capable of hybridizing to the nucleotide sequenceof psaA, such as the psaA nucleotide sequence given below (or thecomplement thereof). An exemplary nucleotide sequence of psaA as foundat GENBANK®Accession number U53509 on May 7, 2007 is shown below:

(SEQ ID NO: 15) tactgcttcagttttgggactctttattggctatagttttaatgttgcggcaggttctagtatcgtgcttacagctgctagtttctttctcattagcttctttatcgctcccaaacaacgatatttgaaactgaaaaataaacatttgttaaaataaggggcaaagccctaataaattggaggatctaatgaaaaaattaggtacattactcgttctctttctttctgcaatcattcttgtagcatgtgctagcggaaaaaaagatacaacttctggtcaaaaactaaaagttgttgctacaaactcaatcatcgctgatattactaaaaatattgctggtgacaaaattgaccttcatagtatcgttccgattgggcaagacccacacgaatacgaaccacttcctgaagacgttaagaaaacttctgaggctgatttgattttctataacggtatcaaccttgaaacaggtggcaatgcttggtttacaaaattggtagaaaatgccaagaaaactgaaaacaaagactacttcgcagtcagcgacggcgttgatgttatctaccttgaaggtcaaaatgaaaaaggaaaagaagacccacacgcttggcttaaccttgaaaacggtattatttttgctaaaaatatcgccaaacaattgagcgccaaagaccctaacaataaagaattctatgaaaaaaatctcaaagaatatactgataagttagacaaacttgataaagaaagtaaggataaatttaataagatccctgctgaaaagaaactcattgtaaccagcgaaggagcattcaaatacttctctaaagcctatggtgtcccaagtgcctacatctgggaaatcaatactgaagaagaaggaactcctgaacaaatcaagaccttggttgaaaaacttcgccaaacaaaagttccatcactctttgtagaatcaagtgtggatgaccgtccaatgaaaactgtttctcaagacacaaacatcccaatctacgcacaaatctttactgactctatcgcagaacaaggtaaagaaggcgacagctactacagcatgatgaaatacaaccttgacaagattgctgaaggattggcaaaataagcctctgaaaaacgtcattctcatgtgagctggcgttttttctatgcccacatttccggtcaaatcattggaaaattctgactgtttcagatacaatggaagaaaaaagattggagtatcctatggtaacttttctcggaaatcctgtgagctttacaggtaaacaactacaagtcggcgacaaggcgcttgatttttctcttactacaaca

TAQMAN® PCR: With reference to FIG. 3, TAQMAN® probe 360 that typicallycontains reporter 320 (such as a short-wavelength fluorophore, forexample 6-carboxyfluorescein (FAM)) and quencher 350 (such as along-wavelength fluorophore, for example BLACKHOLE QUENCHER™ 1 (BHQ™1))is selected to bind to one strand of target nucleic acid 330. Whenirradiated energy is transferred (via FRET) from reporter 320 toquencher 350 on the other end of intact TAQMAN® probe 360. Thus, theclose proximity of reporter 320 and quencher 350 prevents detection ofany signal while TAQMAN® probe 360 is intact. When Taq polymerasereplicates target nucleic acid 330 using primers 300, 301 on whichTAQMAN® probe 360 is bound, polymerase 380's 5′ exonuclease activitycleaves TAQMAN® probe 360. Upon degradation, FRET is interrupted, endingthe activity of quencher 350. Reporter 320 starts to emit signal, whichincreases in each cycle proportional to the rate of TAQMAN® probe 360cleavage. Accumulation of PCR product 370 is detected by monitoring theincrease in signal of reporter 320. Because the cleavage occurs only ifTAQMAN® probe 360 hybridizes to target nucleic acid 330, the origin ofthe detected fluorescence is specific amplification. The process ofhybridization and cleavage does not interfere with the exponentialaccumulation of PCR product 370.

Target nucleic acid molecule: A nucleic acid molecule whose detection,quantitation, qualitative detection, or a combination thereof, isintended. The nucleic acid molecule need not be in a purified form.Various other nucleic acid molecules can also be present with the targetnucleic acid molecule. For example, the target nucleic acid molecule canbe a specific nucleic acid molecule (which can include RNA such as S.pneumoniae RNA, or DNA such as S. pneumoniae DNA, for example a S.pneumoniae lytA, psaA or ply DNA), the amplification of which isintended. Purification or isolation of the target nucleic acid molecule,if needed, can be conducted by methods known to those in the art, suchas by using a commercially available purification kit or the like. Inone example, a target nucleic molecule is a S. pneumoniae nucleic acidsequence.

II. Overview of Several Embodiments

Accurate diagnosis of pneumococcal disease has been frequently hamperednot only by the difficulties in obtaining isolates of the organism frompatient specimens, but also by the misidentification ofpneumococcus-like viridans streptococci species (P-LVS) as Streptococcuspneumoniae (S. pneumoniae). In order to achieve an accurate diagnosis,an assay should be both sensitive and highly specific for diagnosis ofthe disease, such as the diagnosis of a S. pneumoniae infection.Disclosed herein are probes and primers designed for detection S.pneumoniae by detecting of specific sequence regions of the S.pneumoniae lytA, ply, and psaA genes, for example in real-time PCRassays, such as in multiplex real-time PCR assays. When used inrepresentative real-time PCR assays, the probes and primers disclosedherein demonstrated both high sensitivity and high specificity for S.pneumoniae and represent a significant advancement over the probes andprimers typically used for the detection of S. pneumoniae, such as theprobes and primers described by Corless et al. (Corless et al., J. Clin.Microbiol. 39:1553-1558, 2001) and McAvin et al. (McAvin et al., J.Clin. Microbiol. 39:3446-3451, 2001).

A direct comparison (using real-time PCR) of the disclosed probes andprimers (lytA-CDC, psaA-CDC, and ply-CDC) with the probes and primersdescribed by Corless et al. and McAvin et al. (lytA-McAvin andply-Corless) over a panel of isolates consisting of: 67 S. pneumoniae(44 different serotypes and 3 non-encapsulated Streptococcus pneumoniaefrom conjunctivitis outbreaks), and 104 non-pneumococcal isolatesdemonstrated that the probes and primers disclosed herein, such as theprobes and primers specific for S. pneumoniae lytA, ply, and psaA, weresuperior in discriminating between S. pneumoniae isolates and non-S.pneumoniae isolates. The disclosed probes, as well as those described byCorless et al. and McAvin et al., detected the 67 S. pneumoniaeisolates. However, the disclosed S. pneumoniae lytA and psaA specificprobes and primers demonstrated superior specificity for S. pneumoniaeover the assays described by Corless et al. and McAvin et al. Forexample, the probes and primers described by Corless et al. and McAvinet al. registered false positives, and could result in a misdiagnosis ofS. pneumoniae in some instances. Both the lytA-CDC and the psaA-CDCreal-time PCR assays were highly specific, showing no amplification withP-LVS isolates. The newly developed methods described herein provideassays with not only high sensitivity but also improve specificity overthose currently in use. The improvement in specificity allows their usewith specimens from non-sterile sites, as well as sterile sites makingthem suitable for both diagnosis and for use in carriage studies.

Both the lytA-CDC and psaA-CDC assays, and particularly the lytA-CDCassay, represent an improvement in specificity over what is currentlyavailable and should therefore be considered as the assays of choice forthe detection of pneumococcal DNA, particularly when upper respiratoryP-LVS might be present in the clinical specimen. Use of the disclosedprobes and primers for the diagnosis of S. pneumoniae will lead to adecrease in misdiagnosis, improve patient management, and improvemonitoring of S. pneumoniae outbreaks. In addition, as demonstrated byexample 5 (see FIG. 5) the disclosed probes and primers are ideal foruse in multiplex PCR assays for simultaneous detection of variouspathogens. The S. pneumoniae specific probes and primers disclosedherein, such as the lytA, psaA, and ply specific primers and probesprovide high specificity in a respiratory platform multiplexed withprimers and probes for detection of other respiratory pathogens. Thispotential for multiplexing and the speed of performance make theseassays beneficial tools for molecular detection and epidemiologiccarriage studies.

Probes and Primers

Probes capable of hybridizing to and detecting the presence of S.pneumoniae nucleic acid molecules, such as S. pneumoniae lytA nucleicacid molecules, S. pneumoniae psaA nucleic acid molecules, or S.pneumoniae ply nucleic acid molecules, are disclosed. In someembodiments, such probes are specific for S. pneumoniae, in that they donot specifically hybridize to sequences from other organisms, such asother bacteria. The disclosed probes are between 20 and 40 nucleotidesin length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28 29, 30, 31, 32,32, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length and are capableof hybridizing to the S. pneumoniae nucleic acid molecule, such as theS. pneumoniae lytA sequence set forth as SEQ ID NO: 13, the S.pneumoniae psaA sequence set forth as SEQ ID NO: 14, or the S.pneumoniae ply sequence set forth as SEQ ID NO: 15. In severalembodiments, a probe is capable of hybridizing under very highstringency conditions to a S. pneumoniae nucleic acid sequence set forthas SEQ ID NO: 13. In other embodiments, a probe is capable ofhybridizing under very high stringency conditions to a S. pneumoniaenucleic acid sequence set forth as SEQ ID NO: 14. In still otherembodiments, a probe is capable of hybridizing under very highstringency conditions to a S. pneumoniae nucleic acid sequence set forthas SEQ ID NO: 15.

In several embodiments, a probe capable of hybridizing to a S.pneumoniae nucleic molecule contains a nucleic acid sequence that is atleast 95% identical, such as at least 96%, at least 97%, at least 98%,at least 99%, or even 100% identical, to the nucleotide sequence setforth as one of GCCGAAAACGCTTGATACAGGGAG (SEQ ID NO: 5),TGCCGAAAACGCTTGATACAGGGAG (SEQ ID NO: 6),CTAGCACATGCTACAAGAATGATTGCAGAAAGAAA (SEQ ID NO: 9), orCTCAAGTTGGAAACCACGAGTAAGAGTGATGAA (SEQ ID NO: 12).

In several embodiments, a probe capable of hybridizing to a S.pneumoniae lytA nucleic molecule contains a nucleic acid sequence thatis at least 95% identical, such as at least 96%, at least 97%, at least98%, at least 99%, or even 100% identical, to the nucleotide sequenceset forth as SEQ ID NO: 5, or SEQ ID NO: 6. In several embodiments, aprobe capable of hybridizing to a S. pneumoniae lytA nucleic acidmolecule consists essentially of a nucleic acid sequence set forth asSEQ ID NO: 5, or SEQ ID NO: 6.

In several embodiments, a probe capable of hybridizing to a S.pneumoniae psaA nucleic molecule contains a nucleic acid sequence thatis at least 95% identical, such as at least 96%, at least 97%, at least98%, at least 99%, or even 100% identical, to the nucleotide sequenceset forth as SEQ ID NO: 9. In several embodiments, a probe capable ofhybridizing to a S. pneumoniae psaA nucleic acid molecule consistsessentially of a nucleic acid sequence set forth as SEQ ID NO: 9.

In several embodiments, a probe capable of hybridizing to a S.pneumoniae ply nucleic molecule contains a nucleic acid sequence that isat least 95% identical, such as at least 96%, at least 97%, at least98%, at least 99%, or even 100% identical, to the nucleotide sequenceset forth as SEQ ID NO: 12. In several embodiments, a probe capable ofhybridizing to a S. pneumoniae ply nucleic acid molecule consistsessentially of a nucleic acid sequence set forth as SEQ ID NO: 12.

In some embodiments, the probe is detectably labeled, either with anisotopic or non-isotopic label, alternatively the target nucleic acid(such as a S. pneumoniae nucleic acid molecule, for example a S.pneumoniae lytA nucleic acid molecule such as set forth as SEQ ID NO: 13or a subsequence thereof, a S. pneumoniae psaA nucleic acid moleculesuch as set forth as SEQ ID NO: 14 or a subsequence thereof, or a S.pneumoniae ply nucleic acid molecule such as set forth as SEQ ID NO: 15or a subsequence thereof) is labeled. Non-isotopic labels can include afluorescent or luminescent molecule, biotin, an enzyme or enzymesubstrate or a chemical. Such labels are preferentially chosen such thatthe hybridization of the probe with target nucleic acid (such as a S.pneumoniae nucleic acid molecule, for example a S. pneumoniae lytA, a S.pneumoniae psaA, or a S. pneumoniae ply nucleic acid molecule orsubsequence thereof) can be detected. In some examples, the probe islabeled with a fluorophore. Examples of suitable fluorophore labels aregiven above. In some examples, the fluorophore is a donor fluorophore.In other examples, the fluorophore is an accepter fluorophore, such as afluorescence quencher. In some examples, the probe includes both a donorfluorophore and an accepter fluorophore, for example a donor fluorophoresuch as a FAM and an acceptor fluorophore such as a BLACK HOLE®quencher. Appropriate donor/acceptor fluorophore pairs can be selectedusing routine methods. In one example, the donor emission wavelength isone that can significantly excite the acceptor, thereby generating adetectable emission from the acceptor. In some examples, the probe ismodified at the 3′-end to prevent extension of the probe by apolymerase.

In particular examples, the acceptor fluorophore (such as a fluorescencequencher) is attached to the 3′ end of the probe and the donorfluorophore is attached to a 5′ end of the probe. In other examples, theacceptor fluorophore (such as a fluorescence quencher) is attached tothe 5′ end of the probe and the donor fluorophore is attached to a 3′end of the probe. In another particular example, the acceptorfluorophore (such as a fluorescence quencher) is attached to a modifiednucleotide (such as a T) and the donor fluorophore is attached to a 5′end of the probe.

Primers capable of hybridizing to and directing the amplification of aS. pneumoniae nucleic acid molecule are disclosed. In some embodiments,such primers are specific for S. pneumoniae, in that they do notspecifically hybridize to nucleic acid sequences from other organisms,such as other bacteria. The primers disclosed herein are between 15 to40 nucleotides in length, such as 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or even40 nucleotides in length.

In several embodiments, a primer is capable of hybridizing under veryhigh stringency conditions to a S. pneumoniae lytA nucleic acidsequence, such as a S. pneumoniae lytA sequence set forth as SEQ ID NO:13, and directing the amplification of the S. pneumoniae lytA nucleicacid molecule, for example amplification of SEQ ID NO: 13 or asubsequence thereof. In several embodiments, a primer is capable ofhybridizing under very high stringency conditions to a S. pneumoniaepsaA nucleic acid sequence, such as a S. pneumoniae psaA sequence setforth as SEQ ID NO: 14, and directing the amplification of the S.pneumoniae psaA nucleic acid molecule, for example amplification of SEQID NO: 14 or a subsequence thereof. In several embodiments, a primer iscapable of hybridizing under very high stringency conditions to a S.pneumoniae ply nucleic acid sequence, such as a S. pneumoniae plysequence set forth as SEQ ID NO: 15, and directing the amplification ofthe S. pneumoniae ply nucleic acid molecule, for example amplificationof SEQ ID NO: 15 or a subsequence thereof.

In several embodiments, a primer capable of hybridizing to and directingthe amplification of a S. pneumoniae nucleic acid molecule contains anucleic acid sequence that is at least 95% identical, such as at least96%, at least 97%, at least 98%, at least 99%, or even 100% identical,to the nucleic acid sequence set forth as

ACGCAATCTAGCAGATGAAGCA, (SEQ ID NO: 3) TCGTGCGTTTTAATTCCAGCT, (SEQ IDNO: 4) GCCCTAATAAATTGGAGGATCTAATGA, (SEQ ID NO: 7)GACCAGAAGTTGTATCTTTTTTTCCG, (SEQ ID NO: 8) GCTTATGGGCGCCAAGTCTA, (SEQ IDNO: 10) or CAAAGCTTCAAAAGCAGCCTC TA. (SEQ ID NO: 11)

In several embodiments, a primer capable of hybridizing to a S.pneumoniae nucleic acid molecule consists essentially of a nucleic acidsequence set forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 10, or SEQ ID NO: 11. In several embodiments, a primercapable of hybridizing to and directing the amplification of a S.pneumoniae lytA nucleic acid molecule contains a nucleic acid sequencethat is at least 95% identical, such as at least 96%, at least 97%, atleast 98%, at least 99%, or even 100% identical, to the nucleic acidsequence set forth as SEQ ID NO: 3 or SEQ ID NO: 4. In severalembodiments, a primer capable of hybridizing to and directing theamplification of a S. pneumoniae lytA nucleic acid molecule consistsessentially of a nucleic acid sequence set forth as SEQ ID NO: 3 or SEQID NO: 4. In several embodiments, a primer capable of hybridizing to anddirecting the amplification of a S. pneumoniae psaA nucleic acidmolecule contains a nucleic acid sequence that is at least 95%identical, such as at least 96%, at least 97%, at least 98%, at least99%, or even 100% identical, to the nucleic acid sequence set forth asSEQ ID NO: 7 or SEQ ID NO: 8. In several embodiments, a primer capableof hybridizing to and directing the amplification of a S. pneumoniaepsaA nucleic acid molecule consists essentially of a nucleic acidsequence set forth as SEQ ID NO: 7 or SEQ ID NO: 8. In severalembodiments, a primer capable of hybridizing to and directing theamplification of a S. pneumoniae ply nucleic acid molecule contains anucleic acid sequence that is at least 95% identical, such as at least96%, at least 97%, at least 98%, at least 99%, or even 100% identical,to the nucleic acid sequence set forth as SEQ ID NO: 10 or SEQ ID NO:11. In several embodiments, a primer capable of hybridizing to anddirecting the amplification of a S. pneumoniae ply nucleic acid moleculeconsists essentially of a nucleic acid sequence set forth as SEQ ID NO:10 or SEQ ID NO: 11.

In certain embodiments, the primers are a set of primers, such as a pairof primers, capable of hybridizing to and amplifying a S. pneumoniaenucleic acid molecule, such as a S. pneumoniae lytA, a S. pneumoniaepsaA, or a S. pneumoniae ply nucleic acid molecule. Such a set ofprimers includes at least one forward primer and a least one reverseprimer, where the primers are specific for the amplification of a S.pneumoniae nucleic acid molecule such as a S. pneumoniae lytA, a S.pneumoniae psaA, or a S. pneumoniae ply nucleic acid molecule. In someembodiments, the set of primers includes at least one pair of primersspecific for the amplification a S. pneumoniae lytA, a S. pneumoniaepsaA, or a S. pneumoniae ply nucleic acid molecule, for example such aset of primers could include a pair of primers for the amplification ofS. pneumoniae lytA, a pair of primers for the amplification of S.pneumoniae psaA, or a pair of primers for the amplification of S.pneumoniae ply, or any combination thereof, such as a pair of primersfor the amplification of S. pneumoniae lytA and a pair of primers forthe amplification of S. pneumoniae psaA, a pair of primers for theamplification of S. pneumoniae lytA and a pair of primers for theamplification of S. pneumoniae ply, a pair of primers for theamplification of S. pneumoniae ply and a pair of primers for theamplification of S. pneumoniae psaA, or even a pair of primers for theamplification of S. pneumoniae lytA, a pair of primers for theamplification of S. pneumoniae psaA, and a pair of primers for theamplification of S. pneumoniae ply.

In some examples, the set of primers includes a pair of primers that isspecific for the amplification of a S. pneumoniae nucleic acid moleculethat includes a portion of the nucleic acid sequence of the S.pneumoniae lytA gene, such as the nucleic acid sequence set forth as SEQID NO: 13. In certain examples, the pair of primers is specific for theamplification of a S. pneumoniae lytA nucleic acid molecule and includesa forward primer at least 95% identical to SEQ ID NO: 3, such as atleast 96%, at least 97%, at least 98%, at least 99%, or even 100%identical to SEQ ID NO: 3, and a reverse primer at least 95% identicalto SEQ ID NO: 4, such as at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identical to SEQ ID NO: 4.

In some examples, the set of primers includes a pair of primers that isspecific for the amplification of a S. pneumoniae nucleic acid moleculethat includes a portion of the nucleic acid sequence of the S.pneumoniae psaA gene, such as the nucleic acid sequence set forth as SEQID NO: 14. In certain examples, the pair of primers is specific for theamplification of a S. pneumoniae lytA nucleic acid molecule and includesa forward primer at least 95% identical to SEQ ID NO: 7, such as atleast 96%, at least 97%, at least 98%, at least 99%, or even 100%identical to SEQ ID NO: 7, and a reverse primer at least 95% identicalto SEQ ID NO: 8, such as at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identical to SEQ ID NO: 8.

In some examples, the set of primers includes a pair of primers that isspecific for the amplification of a S. pneumoniae nucleic acid moleculethat includes a portion of the nucleic acid sequence of the S.pneumoniae ply gene, such as the nucleic acid sequence set forth as SEQID NO: 14. In certain examples, the pair of primers is specific for theamplification of a S. pneumoniae ply nucleic acid molecule and includesa forward primer at least 95% identical to SEQ ID NO: 10, such as atleast 96%, at least 97%, at least 98%, at least 99%, or even 100%identical to SEQ ID NO: 10, and a reverse primer at least 95% identicalto SEQ ID NO: 11, such as at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identical to SEQ ID NO: 11.

Although exemplary probes and primers are provided in SEQ ID NOS:3-12,the primer and/or probe sequence can be varied slightly by moving theprobes a few nucleotides upstream or downstream from the nucleotidepositions that they hybridize to on the S. pneumoniae nucleic moleculeacid, provided that the probe and/or primer is still specific for the S.pneumoniae nucleic acid sequence, for example specific for SEQ ID NO:13, SEQ ID NO: 14, or SEQ ID NO: 15. For example, variations of theprobes and primers disclosed as SEQ ID NOs:3-12 can be made by “sliding”the probes and/or primers a few nucleotides 5′ or 3′ from theirpositions, and that such variation will still be specific for S.pneumoniae lytA, psaA, or ply.

Also provided by the present application are probes and primers thatinclude variations to the nucleotide sequences shown in any of SEQ IDNOs:3-12, as long as such variations permit detection of the S.pneumoniae nucleic acid molecule. For example, a probe or primer canhave at least 95% sequence identity such as at least 96%, at least 97%,at least 98%, at least 99% to a nucleic acid consisting of the sequenceshown in any of SEQ ID NOs:3-12. In such examples, the number ofnucleotides does not change, but the nucleic acid sequence shown in anyof SEQ ID NOs:3-12 can vary at a few nucleotides, such as changes at 1,2, 3, or 4 nucleotides.

The present application also provides probes and primers that areslightly longer or shorter than the nucleotide sequences shown in any ofSEQ ID NOs:3-12, as long as such deletions or additions permit detectionof the desired S. pneumoniae nucleic acid molecule, such as a S.pneumoniae lytA, psaA or ply sequence. For example, a probe can includea few nucleotide deletions or additions at the 5′- or 3′-end of theprobe or primers shown in any of SEQ ID NOs:3-12, such as addition ordeletion of 1, 2, 3, or 4 nucleotides from the 5′- or 3′-end, orcombinations thereof (such as a deletion from one end and an addition tothe other end). In such examples, the number of nucleotides changes.

Detection of S. pneumoniae

A major application of the S. pneumoniae specific primers and probesdisclosed herein is for the detection of S. pneumoniae in a sample, suchas a biological sample obtained from a subject that has or is suspectedof having an S. pneumoniae infection. Thus, the disclosed methods can beused to diagnose if a subject has a S. pneumoniae. Accordingly, methodsfor the detection of S. pneumoniae nucleic acids are disclosed, forexample to determine if a subject is infected with S. pneumoniae.

The methods described herein may be used for any purpose for whichdetection of S. pneumoniae is desirable, including diagnostic andprognostic applications, such as in laboratory and clinical settings.Appropriate samples include any conventional environmental or biologicalsamples, including clinical samples obtained from a human or veterinarysubject. Suitable samples include all biological samples useful fordetection of bacterial infection in subjects, including, but not limitedto, cells, tissues (for example, lung, liver and kidney), bone marrowaspirates, bodily fluids (for example, blood, serum, urine,cerebrospinal fluid, bronchoalveolar levage, tracheal aspirates, sputum,nasopharyngeal aspirates, oropharyngeal aspirates, saliva), eye swabs,cervical swabs, vaginal swabs, rectal swabs, stool, and stoolsuspensions. Other suitable samples include samples obtained from middleear fluids, bronchoalveolar levage, tracheal aspirates, sputum,nasopharyngeal aspirates, oropharyngeal aspirates, or saliva. Standardtechniques for acquisition of such samples are available. See forexample, Schluger et al., J. Exp. Med. 176:1327-33 (1992); Bigby et al.,Am. Rev. Respir. Dis. 133:515-18 (1986); Kovacs et al., NEJM 318:589-93(1988); and Ognibene et al., Am. Rev. Respir. Dis. 129:929-32 (1984).

Detecting a S. pneumoniae nucleic acid in a sample involves contactingthe sample with at least one of the S. pneumoniae specific probesdisclosed herein that is capable of hybridizing to a S. pneumoniaenucleic acid, such as a S. pneumoniae lytA nucleic acid, S. pneumoniaepsaA nucleic acid, or a S. pneumoniae ply nucleic acid, under conditionsof very high stringency (such as a nucleic acid probe capable ofhybridizing under very high stringency conditions to a S. pneumoniaenucleic acid sequence set forth as SEQ ID NO: 13, SEQ ID NO: 14, or SEQID NO: 15, for example a nucleic acid sequence at least 95% identical tothe nucleotide sequence set forth as one of SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 9, or SEQ ID NO: 12, such as a nucleic acid sequenceconsisting substantially of the nucleic acid sequence set forth as oneof SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, or SEQ ID NO: 12), anddetecting hybridization between the S. pneumoniae nucleic acid and theprobe. Detection of hybridization between the probe and the S.pneumoniae nucleic acid indicates the presence of the S. pneumoniaenucleic acid in the sample, for example detection of hybridizationbetween the S. pneumoniae lytA probe and the S. pneumoniae lytA nucleicacid indicates the presence of the S. pneumoniae nucleic acid in thesample, detection of hybridization between the S. pneumoniae psaA probeand the S. pneumoniae psaA nucleic acid indicates the presence of the S.pneumoniae nucleic acid in the sample, and detection of hybridizationbetween the S. pneumoniae ply probe and the S. pneumoniae ply nucleicacid indicates the presence of the S. pneumoniae nucleic acid in thesample.

In some embodiments, S. pneumoniae nucleic acids present in a sample areamplified prior to using a hybridization probe for detection. Forinstance, it can be advantageous to amplify a portion of the S.pneumoniae nucleic acid, and then detect the presence of the amplifiedS. pneumoniae nucleic acid. For example, to increase the number ofnucleic acids that can be detected, thereby increasing the signalobtained. S. pneumoniae specific nucleic acid primers can be used toamplify a region that is at least about 50, at least about 60, at leastabout 70, at least about 80 at least about 90, at least about 100, atleast about 200, or more base pairs in length to produce amplified S.pneumoniae specific nucleic acids.

Detecting the amplified product typically includes the use of labeledprobes that are sufficiently complementary and hybridize to theamplified S. pneumoniae nucleic acid sequence. Thus, the presence,amount, and/or identity of the amplified product can be detected byhybridizing a labeled probe, such as a fluorescently labeled probe,complementary to the amplified product. In one embodiment, the detectionof a target nucleic acid sequence of interest, such as a S. pneumoniaelytA nucleic acid, a S. pneumoniae psaA nucleic acid, or a S. pneumoniaeply nucleic acid, includes the combined use of PCR amplification and alabeled probe such that the product is measured using real-time PCR. Inanother embodiment, the detection of an amplified target nucleic acidsequence of interest includes the transfer of the amplified targetnucleic acid to a solid support, such as a blot, for example a Northernblot, and probing the blot with a probe, for example a labeled probe,that is complementary to the amplified target nucleic acid sequence. Inyet another embodiment, the detection of an amplified target nucleicacid sequence of interest includes the hybridization of a labeledamplified target nucleic acid to probes disclosed herein that arearrayed in a predetermined array with an addressable location and thatare complementary to the amplified target nucleic acid.

Any nucleic acid amplification method can be used to detect the presenceof S. pneumoniae in a sample. In one specific, non-limiting example,polymerase chain reaction (PCR) is used to amplify the S. pneumoniaenucleic acid sequences. In other specific, non-limiting examples,real-time PCR, reverse transcriptase-polymerase chain reaction (RT-PCR),real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR),ligase chain reaction, or transcription-mediated amplification (TMA) isused to amplify the S. pneumoniae nucleic acid. In a specific example,the S. pneumoniae lytA nucleic acid is amplified by real-time PCR, forexample real-time TAQMAN® PCR. Techniques for nucleic acid amplificationare well-known to those of skill in the art.

Typically, at least two primers are utilized in the amplificationreaction, Amplification of the S. pneumoniae nucleic acid involvescontacting the S. pneumoniae nucleic acid with one or more primers thatare capable of hybridizing to and directing the amplification of a S.pneumoniae nucleic acid (such as a primer capable of hybridizing undervery high stringency conditions to a S. pneumoniae nucleic acid sequenceset forth as SEQ NO:13, SEQ ID NO: 14, or SEQ ID NO: 15, for example aprimer that is least 95% identical (such as 100% identical) to thenucleotide sequence set forth as one of SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 11).

In some embodiments, the sample is contacted with a pair of primers thatinclude a forward and reverse primer that both hybridize to a S.pneumoniae lytA nucleic acid, such as a primer that is least 95%identical (such as 100% identical) to the nucleotide sequence set forthas SEQ ID NO: 3 and a primer that is least 95% identical (such as 100%identical) to the nucleotide sequence set forth as SEQ ID NO: 4. In someembodiments, the sample is contacted with a pair of primers that includea forward and reverse primer that both hybridize to a S. pneumoniae psaAnucleic acid, such as a primer that is least 95% identical (such as 100%identical) to the nucleotide sequence set forth as SEQ ID NO: 7 and aprimer that is least 95% identical (such as 100% identical) to thenucleotide sequence set forth as SEQ ID NO: 8. In some embodiments, thesample is contacted with a pair of primers that include a forward andreverse primer that both hybridize to a S. pneumoniae ply nucleic acid,such as a primer that is least 95% identical (such as 100% identical) tothe nucleotide sequence set forth as SEQ ID NO: 10 and a primer that isleast 95% identical (such as 100% identical) to the nucleotide sequenceset forth as SEQ ID NO: 11.

The amplified S. pneumoniae nucleic acid, can be detected in real-time,for example by real-time PCR, in order to determine the presence, and/orthe amount of S. pneumoniae specific nucleic acid in a sample, such asS. pneumoniae lytA, psaA, or ply nucleic acid. In this manner, anamplified nucleic acid sequence, such as an amplified S. pneumoniaelytA, psaA, or ply nucleic acid sequence, can be detected using a probespecific for the product amplified from the S. pneumoniae sequence ofinterest, such as an amplified S. pneumoniae lytA, psaA, or ply nucleicacid sequence.

Real-time PCR monitors the fluorescence emitted during the reaction asan indicator of amplicon production during each PCR cycle as opposed tothe endpoint detection. The real-time progress of the reaction can beviewed in some systems. Typically, real-time PCR uses the detection of afluorescent reporter. Typically, the fluorescent reporter's signalincreases in direct proportion to the amount of PCR product in areaction. By recording the amount of fluorescence emission at eachcycle, it is possible to monitor the PCR reaction during exponentialphase where the first significant increase in the amount of PCR productcorrelates to the initial amount of target template. The higher thestarting copy number of the nucleic acid target, the sooner asignificant increase in fluorescence is observed.

In one embodiment, the fluorescently-labeled probes rely uponfluorescence resonance energy transfer (FRET), or in a change in thefluorescence emission wavelength of a sample, as a method to detecthybridization of a DNA probe to the amplified target nucleic acid inreal-time. For example, FRET that occurs between fluorogenic labels ondifferent probes (for example, using HybProbes) or between a fluorophoreand a non-fluorescent quencher on the same probe (for example, using amolecular beacon or a TAQMAN® probe) can identify a probe thatspecifically hybridizes to the DNA sequence of interest and in this way,using a S. pneumoniae lytA probe, a S. pneumoniae psaA probe, a S.pneumoniae ply probe, can detect the presence, and/or amount of S.pneumoniae in a sample. In some embodiments, the fluorescently-labeledDNA probes used to identify amplification products have spectrallydistinct emission wavelengths, thus allowing them to be distinguishedwithin the same reaction tube, for example in multiplex PCR, for examplea multiplex real-time PCR. In some embodiments, the probes and primersdisclosed herein are used in multiplex real-time PCR. For example,multiplex PCR permits the simultaneous detection of the amplificationproducts of a lytA, psaA, and ply nucleic acid using the disclosedprobes or even an other nucleic acid, such as a control nucleic acid,for example a RNAse P nucleic acid. Using the disclosed primers andprobes, any combination of lytA, psaA, and ply can be detected.

In another embodiment, a melting curve analysis of the amplified targetnucleic acid can be performed subsequent to the amplification process.The T_(m) of a nucleic acid sequence depends on the length of thesequence and its G/C content. Thus, the identification of the T_(m) fora nucleic acid sequence can be used to identify the amplified nucleicacid, for example by using double-stranded DNA binding dye chemistry,which quantitates the amplicon production by the use of a non-sequencespecific fluorescent intercalating agent (such as SYBR-green or ethidiumbromide). SYBR green is a fluorogenic minor groove binding dye thatexhibits little fluorescence when in solution but emits a strongfluorescent signal upon binding to double-stranded DNA. Typically, SYBRgreen is used in singleplex reactions, however when coupled with meltingpoint analysis, it can be used for multiplex reactions.

Any type of thermal cycler apparatus can be used for the amplificationof the S. pneumoniae nucleic acid, such as a lytA, psaA, or ply nucleicacid and/or the determination of hybridization. Examples of suitableapparatuses include a PTC-100® Peltier Thermal Cycler (MJ Research,Inc.; San Francisco, Calif.), a ROBOCYCLER® 40 Temperature Cycler(Stratagene; La Jolla, Calif.), or a GENEAMP® PCR System 9700 (AppliedBiosystems; Foster City, Calif.). For real-time PCR, any type ofreal-time thermocycler apparatus can be used. For example, a BioRadiCycler iQTM, LIGHTCYCLER™ (Roche; Mannheim, Germany), a 7700 SequenceDetector (Perkin Elmer/Applied Biosystems; Foster City, Calif.), ABI™systems such as the 7000, 7500, 7700, or 7900 systems (AppliedBiosystems; Foster City, Calif.), or an MX4000™ MX3000™ or MX3005™(Stratagene; La Jolla, Calif.); DNA Engine Opticon ContinuousFluorescence Detection System (MJ Research); and Cepheid SMARTCYCLER™can by used to amplify nucleic acid sequences in real-time.

In some embodiments, detecting the presence of a S. pneumoniae nucleicacid sequence in a sample includes the extraction of S. pneumoniae DNA.DNA extraction relates to releasing DNA from a latent or inaccessibleform in a cell or sample and allowing the DNA to become freelyavailable. In such a state, it is suitable for effective detectionand/or amplification of the S. pneumoniae nucleic acid. Releasing DNAmay include steps that achieve the disruption of cells. Additionally,extraction of DNA may include steps that achieve at least a partialseparation of the DNA dissolved in an aqueous medium from other cellularcomponents, wherein such components may be either particulate ordissolved.

In some embodiments, detecting the presence of a S. pneumoniae lytAnucleic acid sequence in a sample includes the extraction of S.pneumoniae RNA. RNA extraction relates to releasing RNA from a latent orinaccessible form in a cell or sample and allowing the RNA to becomefreely available. In such a state, it is suitable for effectivedetection and/or amplification of the S. pneumoniae nucleic acid.Releasing RNA may include steps that achieve the disruption of cells.Extraction of RNA is generally carried out under conditions thateffectively exclude or inhibit any ribonuclease activity that may bepresent. Additionally, extraction of RNA may include steps that achieveat least a partial separation of the RNA dissolved in an aqueous mediumfrom other cellular components, wherein such components may be eitherparticulate or dissolved.

One of ordinary skill in the art will know suitable methods forextracting nucleic acids such as RNA and/or DNA from a sample; suchmethods will depend upon, for example, the type of sample in which theS. pneumoniae nucleic acid is found. For example, the nucleic acids maybe extracted using guanidinium isothiocyanate, such as the single-stepisolation by acid guanidinium isothiocyanate-phenol-chloroformextraction of Chomczynski et al. (Anal. Biochem. 162:156-59, 1987). Thesample can be used directly or can be processed, such as by addingsolvents, preservatives, buffers, or other compounds or substances.Nucleic acids can be extracted using standard methods. For instance,rapid nucleic acid preparation can be performed using a commerciallyavailable kit (such as the QIAGEN® DNA Mini kit (QIAGEN®)Roche MagNAPure Compact Nucleic Acid Isolation Kit I or RNEASY® Mini Kit (QIAGEN®);NUCLISENS® NASBA Diagnostics (bioMérieux); or the MASTERPURE™ CompleteDNA and RNA Purification Kit (EPICENTRE)).

In some embodiments, the probe is detectably labeled, either with anisotopic or non-isotopic label; in alternative embodiments, the S.pneumoniae nucleic acid is labeled. Non-isotopic labels can, forinstance, comprise a fluorescent or luminescent molecule, or an enzyme,co-factor, enzyme substrate, or hapten. The probe is incubated with asingle-stranded or double-stranded preparation of RNA, DNA, or a mixtureof both, and hybridization determined. In some examples, thehybridization results in a detectable change in signal such as inincrease or decrease in signal, for example from the labeled probe.Thus, detecting hybridization comprises detecting a change in signalfrom the labeled probe during or after hybridization relative to signalfrom the label before hybridization.

Streptococcus pneumoniae Identification Arrays

An array containing a plurality of heterogeneous probes for thedetection, of S. pneumoniae are disclosed. Such arrays may be used torapidly detect S. pneumoniae in a sample.

Arrays are arrangements of addressable locations on a substrate, witheach address containing a nucleic acid, such as a probe, such as a S.pneumoniae lytA psaA, or ply probe as disclosed herein. In someembodiments, each address corresponds to a single type or class ofnucleic acid, such as a single probe, though a particular nucleic acidmay be redundantly contained at multiple addresses. A “microarray” is aminiaturized array requiring microscopic examination for detection ofhybridization. Larger “macroarrays” allow each address to berecognizable by the naked human eye and, in some embodiments, ahybridization signal is detectable without additional magnification. Theaddresses may be labeled, keyed to a separate guide, or otherwiseidentified by location.

In some embodiments, a S. pneumoniae detection array is a collection ofseparate probes at the array addresses. The S. pneumoniae detectionarray is then contacted with a sample suspected of containing S.pneumoniae nucleic acids under conditions allowing hybridization betweenthe probe and nucleic acids in the sample to occur. Any samplepotentially containing, or even suspected of containing, S. pneumoniaenucleic acids may be used, including nucleic acid extracts, such asamplified or non-amplified DNA or RNA preparations. A hybridizationsignal from an individual address on the array indicates that the probehybridizes to a nucleotide within the sample. This system permits thesimultaneous analysis of a sample by plural probes and yieldsinformation identifying the S. pneumoniae nucleic acids contained withinthe sample. In alternative embodiments, the array contains S. pneumoniaenucleic acids and the array is contacted with a sample containing aprobe. In any such embodiment, either the probe or the S. pneumoniaenucleic acids may be labeled to facilitate detection of hybridization.

The nucleic acids may be added to an array substrate in dry or liquidform. Other compounds or substances may be added to the array as well,such as buffers, stabilizers, reagents for detecting hybridizationsignal, emulsifying agents, or preservatives.

In certain examples, the array includes one or more molecules or samplesoccurring on the array a plurality of times (twice or more) to providean added feature to the array, such as redundant activity or to provideinternal controls.

Within an array, each arrayed nucleic acid is addressable, such that itslocation may be reliably and consistently determined within the at leastthe two dimensions of the array surface. Thus, ordered arrays allowassignment of the location of each nucleic acid at the time it is placedwithin the array. Usually, an array map or key is provided to correlateeach address with the appropriate nucleic acid. Ordered arrays are oftenarranged in a symmetrical grid pattern, but nucleic acids could bearranged in other patterns (for example, in radially distributed lines,a “spokes and wheel” pattern, or ordered clusters). Addressable arrayscan be computer readable; a computer can be programmed to correlate aparticular address on the array with information about the sample atthat position, such as hybridization or binding data, including signalintensity. In some exemplary computer readable formats, the individualsamples or molecules in the array are arranged regularly (for example,in a Cartesian grid pattern), which can be correlated to addressinformation by a computer.

An address within the array may be of any suitable shape and size. Insome embodiments, the nucleic acids are suspended in a liquid medium andcontained within square or rectangular wells on the array substrate.However, the nucleic acids may be contained in regions that areessentially triangular, oval, circular, or irregular. The overall shapeof the array itself also may vary, though in some embodiments it issubstantially flat and rectangular or square in shape.

S. pneumoniae detection arrays may vary in structure, composition, andintended functionality, and may be based on either a macroarray or amacroarray format, or a combination thereof. Such arrays can include,for example, at least 10, at least 25, at least 50, at least 100, ormore addresses, usually with a single type of nucleic acid at eachaddress. In the case of macroarrays, sophisticated equipment is usuallynot required to detect a hybridization signal on the array, thoughquantification may be assisted by standard scanning and/orquantification techniques and equipment. Thus, macroarray analysis asdescribed herein can be carried out in most hospitals, agricultural andmedial research laboratories, universities, or other institutionswithout the need for investment in specialized and expensive readingequipment.

Examples of substrates for the arrays disclosed herein include glass(e.g., functionalized glass), Si, Ge, GaAs, GaP, SiO₂, SiN₄, modifiedsilicon nitrocellulose, polyvinylidene fluoride, polystyrene,polytetrafluoroethylene, polycarbonate, nylon, fiber, or combinationsthereof. Array substrates can be stiff and relatively inflexible (forexample glass or a supported membrane) or flexible (such as a polymermembrane). One commercially available product line suitable for probearrays described herein is the Microlite line of MICROTITER® platesavailable from Dynex Technologies UK (Middlesex, United Kingdom), suchas the Microlite 1+96-well plate, or the 384 Microlite+384-well plate.

Addresses on the array should be discrete, in that hybridization signalsfrom individual addresses can be distinguished from signals ofneighboring addresses, either by the naked eye (macroarrays) or byscanning or reading by a piece of equipment or with the assistance of amicroscope (microarrays).

Addresses in an array may be of a relatively large size, such as largeenough to permit detection of a hybridization signal without theassistance of a microscope or other equipment. Thus, addresses may be assmall as about 0.1 mm across, with a separation of about the samedistance. Alternatively, addresses may be about 0.5, 1, 2, 3, 5, 7, or10 mm across, with a separation of a similar or different distance.Larger addresses (larger than 10 mm across) are employed in certainembodiments. The overall size of the array is generally correlated withsize of the addresses (for example, larger addresses will usually befound on larger arrays, while smaller addresses may be found on smallerarrays). Such a correlation is not necessary, however.

The arrays herein may be described by their densities (the number ofaddresses in a certain specified surface area). For macroarrays, arraydensity may be about one address per square decimeter (or one address ina 10 cm by 10 cm region of the array substrate) to about 50 addressesper square centimeter (50 targets within a 1 cm by 1 cm region of thesubstrate). For microarrays, array density will usually be one or moreaddresses per square centimeter, for instance, about 50, about 100,about 200, about 300, about 400, about 500, about 1000, about 1500,about 2,500, or more addresses per square centimeter.

The use of the term “array” includes the arrays found in DNA microchiptechnology. As one, non-limiting example, the probes could be containedon a DNA microchip similar to the GENECHIP® products and relatedproducts commercially available from Affymetrix, Inc. (Santa Clara,Calif.). Briefly, a DNA microchip is a miniaturized, high-density arrayof probes on a glass wafer substrate. Particular probes are selected,and photolithographic masks are designed for use in a process based onsolid-phase chemical synthesis and photolithographic fabricationtechniques similar to those used in the semiconductor industry. Themasks are used to isolate chip exposure sites, and probes are chemicallysynthesized at these sites, with each probe in an identified locationwithin the array. After fabrication, the array is ready forhybridization. The probe or the nucleic acid within the sample may belabeled, such as with a fluorescent label and, after hybridization, thehybridization signals may be detected and analyzed.

Kits

The nucleic acid primers and probes disclosed herein can be supplied inthe form of a kit for use in the detection S. pneumoniae, including kitsfor any of the arrays described above. In such a kit, an appropriateamount of one or more of the nucleic acid probes and/or primers (such asS. pneumoniae lytA psaA, or ply probes and primers as disclosed herein)is provided in one or more containers or held on a substrate. A nucleicacid probe and/or primer may be provided suspended in an aqueoussolution or as a freeze-dried or lyophilized powder, for instance. Thecontainer(s) in which the nucleic acid(s) are supplied can be anyconventional container that is capable of holding the supplied form, forinstance, microfuge tubes, ampoules, or bottles. The kits can includeeither labeled or unlabeled nucleic acid probes for use in detection ofS. pneumoniae nucleotide sequences.

In some applications, one or more primers (as described above), such aspairs of primers, may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers. Withsuch an arrangement, the sample to be tested for the presence of S.pneumoniae nucleic acids can be added to the individual tubes andamplification carried out directly.

The amount of nucleic acid primer supplied in the kit can be anyappropriate amount, and may depend on the target market to which theproduct is directed. For instance, if the kit is adapted for research orclinical use, the amount of each nucleic acid primer provided wouldlikely be an amount sufficient to prime several PCR amplificationreactions. General guidelines for determining appropriate amounts may befound in Innis et al., Sambrook et al., and Ausubel et al. A kit mayinclude more than two primers in order to facilitate the PCRamplification of a larger number of S. pneumoniae nucleotide sequences.

In some embodiments, kits also may include the reagents necessary tocarry out PCR amplification reactions, including DNA sample preparationreagents, appropriate buffers (such as polymerase buffer), salts (forexample, magnesium chloride), and deoxyribonucleotides (dNTPs).

One or more control sequences for use in the PCR reactions also may besupplied in the kit (for example, for the detection of human RNAse P).

Particular embodiments include a kit for detecting a S. pneumoniaenucleic acid based on the arrays described above. Such a kit includes atleast one probe specific for a S. pneumoniae nucleic acid (as describedabove) and instructions. A kit may contain more than one differentprobe, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,50, 100, or more probes. The instructions may include directions forobtaining a sample, processing the sample, preparing the probes, and/orcontacting each probe with an aliquot of the sample. In certainembodiments, the kit includes an apparatus for separating the differentprobes, such as individual containers (for example, microtubules) or anarray substrate (such as, a 96-well or 384-well microtiter plate). Inparticular embodiments, the kit includes prepackaged probes, such asprobes suspended in suitable medium in individual containers (forexample, individually sealed EPPENDORF® tubes) or the wells of an arraysubstrate (for example, a 96-well microtiter plate sealed with aprotective plastic film). In other particular embodiments, the kitincludes equipment, reagents, and instructions for extracting and/orpurifying nucleotides from a sample.

Synthesis of Oligonucleotide Primers and Probes

In vitro methods for the synthesis of oligonucleotides are well known tothose of ordinary skill in the art; such methods can be used to produceprimers and probes for the disclosed methods. The most common method forin vitro oligonucleotide synthesis is the phosphoramidite method,formulated by Letsinger and further developed by Caruthers (Caruthers etal., Chemical synthesis of deoxyoligonucleotides, in Methods Enzymol.154:287-313, 1987). This is a non-aqueous, solid phase reaction carriedout in a stepwise manner, wherein a single nucleotide (or modifiednucleotide) is added to a growing oligonucleotide. The individualnucleotides are added in the form of reactive 3′-phosphoramiditederivatives. See also, Gait (Ed.), Oligonucleotide Synthesis. Apractical approach, IRL Press, 1984.

In general, the synthesis reactions proceed as follows: Adimethoxytrityl or equivalent protecting group at the 5′ end of thegrowing oligonucleotide chain is removed by acid treatment. (The growingchain is anchored by its 3′ end to a solid support such as a siliconbead.) The newly liberated 5′ end of the oligonucleotide chain iscoupled to the 3′-phosphoramidite derivative of the next deoxynucleotideto be added to the chain, using the coupling agent tetrazole. Thecoupling reaction usually proceeds at an efficiency of approximately99%; any remaining unreacted 5′ ends are capped by acetylation so as toblock extension in subsequent couplings. Finally, the phosphite triestergroup produced by the coupling step is oxidized to the phosphotriester,yielding a chain that has been lengthened by one nucleotide residue.This process is repeated, adding one residue per cycle. See, forexample, U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679, and5,132,418. Oligonucleotide synthesizers that employ this or similarmethods are available commercially (for example, the PolyPlexoligonucleotide synthesizer from Gene Machines, San Carlos, Calif.). Inaddition, many companies will perform such synthesis (for example,Sigma-Genosys, The Woodlands, Tex.; Qiagen Operon, Alameda, Calif.;Integrated DNA Technologies, Coralville, Iowa; and TriLinkBioTechnologies, San Diego, Calif.).

The following examples are provided to illustrate particular features ofcertain embodiments. However, the particular features described belowshould not be construed as limitations on the scope of the invention,but rather as examples from which equivalents will be recognized bythose of ordinary skill in the art.

EXAMPLES Example 1 Materials and Methods

This example describes the materials and methods used to determine thespecificity and sensitivity of the disclosed probes and primers.

Bacterial Isolates

The disclosed probes and primers where tested for specificity for S.pneumoniae using a panel that included 67 S. pneumoniae strainsrepresenting 44 different S. pneumoniae serotypes (1, 2, 4, 5, 6A, 6B,7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 13, 14, 15B, 15A, 15C, 16F, 17A,17F, 18B, 18C, 18F, 19A, 19C, 19F, 20, 21, 22A, 22F, 23B, 23F, 24A, 24B,28A, 28F, 32F, 33A, 33F, 35A, 35F, 40, and 3 non-capsulated fromconjunctivitis outbreaks) and 104 non-pneumococcal isolates including S.pseudopneumoniae, S. mitis, S. oxalis, S. sanguinis, S. parasanguinis,S. peroris, S. infantis, S. gordonii, S. cristatus, S. salivarius, S.vestibularis, S. australis, S. sinensis, S. oligofermentans, S.intestinalis, S. pyogenes, S. agalactiae, S. canis, S. anginosus, S.equi subsp. equi, S. equi subsp. zooepidemicus, S. porcinus, S.dysgalactiae, S. constellatus, S. iniae, S. intermedius, S. aureus, S.warneri, 13 viridans streptococci not identified to the species level,Dolostigranulum pigrum, Enterococcus faecalis, Escherichia coli,Chlamydia pneumoniae, C. psittaci, Mycoplasma pneumoniae, Legionellapneumophila, Haemophilus influenzae types a-f and NT, H. parainfluenzae,Corynebacterium diphtheriae, C. pseudotuberculosis, Nocardia farcinica,N. asteroides, Klebsiella pneumoniae, Mycobaterium fortuitum, M.tuberculosis, Pseudomonas aeruginosa, Bordetella pertussis and B.bronchiseptica.

S. pneumoniae ATCC strain 33400 was used as a positive control in allassays described below. All bacteria isolates were obtained from theculture collections of CDC laboratories (Streptococcus Laboratory,Respiratory Diseases Branch and Molecular Sequencing Laboratory,Meningitis and Vaccine Preventable Diseases Branch).

Optochin Susceptibility Test (OPT)

OPT susceptibility testing was performed on 5% sheep blood agar platesat 5% CO₂ environments as described in Arbique et al. (Arbique et al.,J. Clin. Microbiol. 42: 4686-4696, 2004, which is incorporated herein byreference to the extent that it discloses this test).

Bile Solubility (BS) Test

The tube BS test was performed as previously described (Arbique et al.,J. Clin. Microbiol. 42: 4686-4696, 2004, and Ruoff et al., Streptococcusp. 405-421. In P. R. Murray et al. (ed.), Manual of clinicalmicrobiology 8th ed. American Society for Microbiology, Washington, D.C,2003, both of which is incorporated herein by reference to the extentthat they discloses this test).

DNA Probe Hybridization Test

The ACCUPROBE® Streptococcus pneumoniae culture identification test,based on the rRNA gene sequence, was performed according to themanufacturer's instructions (Gen-Probe, San Diego, Calif.).

DNA-DNA Reassociation

Growth, harvesting and lysis of the bacterial cells were performed asdescribed previously (Arbique et al., J. Clin. Microbiol. 42: 4686-4696,2004, and Brenner et al., J. Clin. Microbiol. 15:1133-1140, 1982).Extraction and purification of DNA and DNA-DNA reassociation studiesincluding determination of DNA relatedness by the hydroxyapatitehybridization method were performed as described by Brenner andcolleagues (Brenner et al., J. Clin. Microbiol. 15:1133-1140, 1982). DNAhybridization experiments were performed at 55° C. for optimal DNAreassociation and at the stringent DNA reassociation temperature of 70°C. The levels of divergence within related sequences were determined byassuming that each degree of heteroduplex instability was caused by 1%unpaired bases. Divergence, expressed by the change in meltingtemperature, is the decrease in the thermal stability (in degreesCelsius) of the heterologous DNA duplex relative to that of thehomologous duplexes. Divergence was calculated to the nearest 0.5%.

Clinical Specimens

Clinical specimens included serum, middle ear fluids (MEFs), andcerebral spinal fluids (CSFs) and were obtained in accordance with CDCInstitutional Review Board (IRB) stipulations. The sera and MEFs wereobtained from the Soroka University Hospital in Beer-Sheva, Israel.Serum specimens were collected from 15 patients with pneumococcalbacteremia and 15 age-matched, ethnic group-matched, healthy controlchildren in whom nasopharyngeal (NP) culture was negative for S.pneumoniae. MEF specimens consisted of 10 S. pneumoniae culture positivemiddle ear fluids and 10 S. pneumoniae culture negative but H.influenzae positive MEFs. Twenty-five CSFs were obtained from theLaboratorio Central do Estado do Rio Grande do Sul, Porto Alegre, Brasiland consisted of 15 specimens from meningitis patients that werepneumococcal culture positive and 10 CSFs from pneumococcal negative, N.meningitidis positive patients. Specimens were shipped on dry ice andfrozen at −70° C. upon arrival.

DNA Extraction for Real-Time PCR Analysis

DNA was extracted from the isolates by a modification of the QIAGEN® DNAMini kit (QIAGEN® Inc., Valencia, Calif.) method. Briefly, a loopfull ofovernight growth from a blood agar plate was resuspended in lysis buffer(20 mM Tris-HCL pH 8.0, 2.0 mM EDTA, 1.2% Triton X100 containing 0.04g/ml lysozyme and 75 U/ml of mutanolysin (Sigma Chemical Co, St Louis,Mo.) and incubated for one hour at 37° C. in a water-bath. The remainingprocedures followed the manufacture's recommendations.

For clinical specimens, 200 μl of clinical material was added to 100 μlof TE buffer containing 0.04 g/ml lysozyme and 75 U/ml of mutanolysin(Sigma Chemical Co) and incubated for one hour in a 37° C. water bath.All subsequent steps were as outlined in the QIAGEN® DNA mini protocolbooklet. DNA was eluted in 100 μl of QIAGEN® EA elution buffer andstored at −20° C. Concentrations of extracted DNA from bacterialcultures were determined by NANODROP® (NANODROP® Technologies,Wilmington, Del.).

Real-Time PCRs for lytA, ply and psaA

The two previously published real-time PCR assays were performed asdescribed (Corless et al., J. Clin. Microbiol. 39:1553-1558, 2001;McAvin et al., J. Clin. Microbiol. 39:3446-3451, 2001).

For development of the assays disclosed herein, oligonucleotide primersand fluorescent dye-labeled probes were designed based on previouslypublished lytA, ply, and psaA gene sequences and sequences available inGENBANK® using the PRIMER EXPRESS® Software (Applied Biosystems, AB,Foster City, Calif.). The probes were labeled 5′ with either6-carboxy-fluorescein (FAM) or in the case of the psaA probe withhexachloro-6-carboxy-fluorescein (HEX). Black hole quencher (BHQ1®,Biosearch Technologies, Novato, Calif.) was either placed at the 3′ endof the probe or internally on a thymidine (Table 1). If internallyquenched the 3′ end was capped with a phosphate group to preventextension of the probe. Primer and probe sequences are listed in Table1.

TABLE 1 Real-time PCR primers and probes Nucleotide AccessionOligonucleotide Sequence position number lytA-CDC forward5′-ACGCAATCTAGCAGATGAAGCA-3′ 1841014 AE005672 (SEQ ID NO: 3) lytA-CDCreverse 5′-TCGTGCGTTTTAATTCCAGCT-3′ 1840961 (SEQ ID NO: 4) lytA-CDC_1probe 5′-FAM GCCGAAAACGCTTGATACAGGGAG-3′ 1840985 BHQ1 (SEQ ID NO: 5)lytA-CDC_2 probe 5′-FAM TGCCGAAAACGCTTGATACAGGGAG-3′ 1840984 BHQ1 (SEQID NO: 6) psaA-CDC forward 5′-GCCCTAATAAATTGGAGGATCTAATGA-3′ 166 U53509(SEQ ID NO: 7) psaA-CDC reverse 5′-GACCAGAAGTTGTATCTTTTTTTCCG-3′ 279(SEQ ID NO: 8) psaA-CDC probea,b 5′-HEX CTAGCACATGCTACAAGAATGATTGC 219AGAAAGAAA-3′ phosphate (SEQ ID NO: 9) ply-CDC forward5′-GCTTATGGGCGCCAAGTCTA -3′ 721 AE008539 (SEQ ID NO: 10) ply-CDC reverse5′-CAAAGCTTCAAAAGCAGCCTC TA-3′ 798 (SEQ ID NO: 11) ply-CDC probeb,c5′-FAM CTCAAGTTGGAAACCACGAGTAAGA 742 GTGATGAA-3′ phosphate (SEQ ID NO:12) ^(a,)psaA probe is designed to bind to the reverse strand of theamplicon ^(b,)T indicates the thymidine on which the internal BHQquencher was attached ^(c,)For multiplex detection the 5′end label waschanged to CAL Flour 61Assays were carried out in a final 25 μl reaction volume, using theTAQMAN®Universal Master Mix kit (AB) according to instructions with 2.5μl of sample DNA. Primer and probe concentrations for each of the threeassays were optimized; and in accordance with the experimentallyoptimized concentrations, 500, 200, 200 nM of psaA-CDC, lytA-CDC,ply-CDC primers and 100, 200, 200 nM of psaA-CDC, lytA-CDC, ply-CDCprobes, respectively, were used for subsequent experiments. Ano-template control and a Streptococcus pneumoniae positive DNA control(S. pneumoniae ATCC 33400) were included in every run. DNA was amplifiedwith Mx3000P (Stratagene, La Jolla, Calif.) or 7500 Real-time PCR system(AB), using the following cycling parameters: 95° C. for 10 min followedby 40 cycles of 95° C. for 15 s and 60° C. for 1 min. Amplification datawere analyzed by instrument software (STRATAGENE® or AppliedBiosystems). Negative samples were defined as those with cyclethresholds greater than >40. New assays are designated lytA-CDC,ply-CDC, and psaA-CDC.

Real-Time PCR Analytical Sensitivity and Specificity Determinations

For lower limit of detection (LLD) assessments, serial 10-fold dilutions(equivalent to 6666 to 6.6 copies) of purified DNA from the pneumococcalreference strain ATCC 33400 were prepared and aliquots tested using allfive real-time PCR protocols. Specificity determinations were made bytesting extracted DNAs at 5 ng/ul from 67 S. pneumoniae isolates and 104non pneumococcal isolates (listed above) in all five assays.

Real-Time PCR of Clinical Samples

S. pneumoniae DNA detection in serum, MEFs and CSFs were performed inparallel on aliquots of the same specimen for all assays. Extracted DNA(2.50 μl of undiluted or 2.50 of 1:3 dilution) from serum, MEFs or CSFwas used in amplification reactions. All assays of each clinical samplewere performed in triplicate. A specimen was considered positive if twoof the three triplicates gave a positive result within the <40 cyclecut-off. Assay protocols were as described above. An RNaseP human genecontrol reaction was performed independently on each sample to check forthe presence of inhibitors. Failure to get amplification in thisreaction was considered indicative of inhibitors.

Mutiplex Real-Time PCRs for psaA-CDC, lytA-CDC and ply-CDC

The three sets of primers and probes were combined into a singlereaction mixture for multiplex detection. Modifications to the singlegene detection assays included: use of QIAGEN's® QUANTITECT® MultiplexPCR NoROX master mix, changing the ply-CDC gene probe fluorescent labelfrom FAM to 5′ CAL FLOUR RED 610®, 3′ BHQ2® (Biosearch Technologies) andreduction of the concentration of the lytA FAM probe to 100 nM from theoriginal to 200 nM. Temperatures and number of cycles remained the sameas described in the original single PCR protocols.

Example 2 Lower Limit of Detection of Assays for Streptococcuspneumoniae Detection

This example describes the methodology employed to determine thesensitivity of the disclosed probes and primers and methods of usingsuch probes and primers versus the probe and primer sets described byCorless et al. and McAvin et al.

The analytical lower limit of detection (LLD) for the five assays weremeasured by amplifying serial dilutions of purified extracted genomicDNA from the positive control strain Streptococcus pneumoniae ATCC33400. All five assays showed a high sensitivity with their respectiveprimer pairs and probes with a limit of detection equivalent to <10copies, except for the psaA-CDC that was approximately 2 fold lesssensitive. All standard curves generated had slopes of −3.4 to −3.2 withR²>0.96. Efficiencies of the assays were very similar ranging from 96%to 100%. Evaluation of the five assays for their ability to amplify DNAfrom a panel of 67 S. pneumoniae strains representing 45 serotypes andnon-typeable S. pneumoniae resulted in 100% amplification or detectionof DNA from all S. pneumoniae strains tested.

Example 3 Specificity of Assays for Streptococcus pneumoniae Detection

This example describes the methodology employed to determine thespecificity of the disclosed probes and primers and methods of usingsuch probes and primers versus the probe and primer sets described byCorless et al. and McAvin et al.

The analytical specificity of each of the five assays was evaluated andcompared by amplifying extracted DNA from 104 strains ofnon-pneumococcal bacteria. These strains represented several genera ofgram-positive and gram-negative bacteria some of which inhabit the oralcavity. No amplification occurred with any of the non streptococci inthe specificity panel. There was however amplification with some strainsof P-LVS and Spseudo. The P-LVS were specifically selected from amongstrains that were submitted to the Streptococcus Laboratory, which hadbeen difficult to identify or classify using the standard methodologycriteria. DNA/DNA reassociation analysis had been performed on theseisolates in addition to BS, OPT, and ACCUPROBE®. DNA/DNA reassociationvalues revealed that these P-LVS and the Spseudo (Table 2) were not S.pneumoniae.

TABLE 2 DNA-DNA hybridization and real-time PCR for un-identifiedviridans streptococci and Streptococcus pseudopneumoniae Referencestrains DNA labeled: S. pneumoniae ATCC 33400^(T)/ Identification testS. pseudopneumoniae ATCC BAA 960^(T) Real-time PCR Geographic RBR at RBRat psaA- psaA- lytA- ply- ply- Strains Origin Specimen OPT BS GP 55° C.70° C. D CDC McAvin CDC Corless CDC S. pneumoniae ATCC 33400^(T) S + +100/55  100/28  0.0/3.5 + + + + + Un-identified viridans streptococci868-84 MD blood R − − 66/nd 59/nd 4.5/nd  − − − + + 2901-90 AL throatR + − 65/nd 51/nd 6.0/nd  − − − + + 2904-90 AL throat S + − 61/nd 49/nd5.5/nd  − − − + − 2909-90 AL throat R − − 61/nd 46/nd 6.0/nd  − − − + +2913-90 AL throat S − − 65/52 58/44 4.5/4.5 − − − + + 2916-90 AL throatR − − 58/nd 42/nd 6.0/nd  − − − + + 2918-90 AL throat S + − 63/nd 54/nd6.0/nd  − − − + + 2919-90 AL throat R − − 63/nd 54/nd 4.5/nd  − − − + −2920-90 AL throat S + − 62/nd 53/nd 6.5/nd  − − − + + 2921-90 AL throatR − − 62/nd 58/nd 4.5/nd  − − − + − 2939-90 AL throat R − − 66/nd 57/nd4.0/nd  − − − + + S. pseudopneumoniae ATCC BAA-960^(T) NS-CA sputum R− +  62/100  56/100 4.0/0.0 − − − + + 253-03 NS-CA sputum R − + 70/8460/74 4.0/2.0 − − − + + 276-03 NS-CA sputum R − + 68/83 53/77 4.0/1.0 −− − + + 288-03 NS-CA sputum R − + 70/82 57/50 4.0/0.5 + + − + + 290-03NS-CA sputum R − + 70/81 64/75 4.0/1.5 − − − + + 2482-91 AL throat R − +58/72 46/71 3.0/1.0 − − − + + 2483-91 AL throat R − + 58/76 46/713.5/1.5 − − − + + 2497-91 AL throat R − + 37/82 49/80 3.5/2.0 + − − + +2946-98 AL throat R − + 61/76 54/76 3.0/1.5 − + − + + 2987-98 AZ NP swabR − + 61/98 50/73 3.0/1.0 − + − + +^(T), type strain; OPT, susceptibility to optochin; BS, bile solubility;GP, GenProbe Accuprobe Pneumococcus culture identification test; RBRrelative binding relation, at 55° C. (optimal temperature) at 70° C.(stringent temperature); D, divergence calculated to the nearest 0.5%;nd, not done; AL, Alaska; AZ, Arizona; MD, Maryland; NS-CA, NovaScotia-Canada;Analysis of these strains using real-time PCR demonstrated that the newlytA-CDC real-time PCR assay was the most specific (100%), showing nodetectable fluorescent signal with DNA from non-S. pneumoniae organismsin the specificity panel (Table 2). This was followed by psaA-CDCreal-time PCR (98%), which gave positive results with two of the Spseudoand the psaA-McAvin real-time PCR (96%) published by McAvin et al. whichwas positive for four Spseudo. No amplification occurred with DNAs fromP-LVS with psaA-McAvin, lytA-CDC and psaA-CDC primer probe sets (Table2). The two assays for the ply S. pneumoniae gene gave positivereactions with all Spseudo; of the other P-LVS, positive reactionsoccurred with both ply (13 of 13) and ply-CDC (10 of 13) assays, makingfinal specificities 78% and 81% respectively.

Both the lytA-CDC and the psaA-CDC real-time PCR were highly specific,showing no amplification with P-LVS isolates. The psaA-CDC real-time PCRwas slightly less specific, amplifying two of the Spseudo. These resultscorrelate with an earlier study using conventional PCR, showing theutility of these genes in discriminating S. pneumoniae.

Example 4 Detection of Streptococcus pneumoniae in Clinical Samples

This example describes the detection of S. pneumoniae in clinicalsamples using the disclosed probes and primers and methods of using suchprobes and primers versus the probe and primer sets described by Corlesset al. and McAvin et al.

The five assays were used on three types of clinical specimens(described above) to evaluate and compare assays (Table 3).

TABLE 3 Assay results for clinical specimens for all five real-timePCRs. Real-time-PCR S. pneumoniae psaA-CDC psaA-McAvin lytA-CDC ply-CDCply-Corless Specimens^(a) Culture N. (+) (−) (+) (−) (+) (−) (+) (−) (+)(−) Serum +  15^(b) 7 8 7 8   8^(c) 7   8^(c) 7 6 9 − 15 0 15 0 15  1^(c) 14   1^(c) 14 0 15 MEF + 10 10  0 10  0 10 0 10 0 10  0 − 10 6 46 4  6 4   7^(c) 3 6 4 CSF + 15 15  0 15  0 15 0 15 0 15  0 − 10  1^(d)9   2^(c,d) 8   3^(c,d) 7   2^(c,d) 8  2^(d) 8 ^(a)MEF, middle earfluids; CSF, cerebral spinal fluids ^(b)One serum was RNase P genenegative ^(c)Average of Ct values = ≦38 for psaA-McAvin, lytA-CDC,ply-CDC ^(d)Ct values ≦37 for all 5 PCR tested for that one specimen

All five assays showed excellent correlation. When specimens werepositive or negative in one assay, identical specimens were generallypositive or negative in the other assays. Differences were only in totalnumbers positive or negative for each. Sensitivities were calculatedbased on the 15 culture positive serum specimens even though one ofthese was RNase P negative, indicating inhibition. Sensitivities withserum samples were 53% (8/15) for lytA-CDC and ply-CDC (1 additionalpositive for each but different specimens), 47% (7/15) for lytA andpsaA, and 40% (6/15) for S. pneumoniae ply. Analysis of the S.pneumoniae culture negative sera showed good correlation between theassays as well as good specificities. No positives occurred for psaA,lytA and ply, resulting in a 100% specificity with serum.

Analysis of MEFs and CSFs revealed that all five assays gave positiveresults for all 10 of the Spn-positive MEFs and all 15 CSFs, yieldingsensitivities of 100%. Specificity evaluations of the 10 culturenegative MEFs resulted in positives with the same six MEF specimens forpsaA-CDC, psaA-McAvin, lytA-CDC, and ply-Corless real-time PCR assaysyielding 40% specificities. An additional specimen was positive byply-CDC for a 30% specificity. Examination of Streptococcuspneumoniae-negative CSFs showed good specificity. Positives were 1 of 10(90% specificity) for psaA, 2 of 10 (80% specificity) for psaA-McAvin,ply-CDC, ply-Corless and 3 of 10 (70% specificity) for lytA-CDC.

Example 5 Mutiplex CDC Real-Time PCRs for psaA, lytA, and ply

This example describes the detection of S. pneumoniae using thedisclosed probes and primers in a multiplex real-time PCR assay.

To ascertain if combining the primer probe sets to detect all threegenes at once would be advantageous in improving sensitivity weconstructed a multiplex of the three newly developed assays. LLDevaluations with serial dilutions of known concentrations of thepneumococcal positive control ATCC 33400 were done and results comparedto the singleplex assay for each gene. These studies showed thatvariation was less than ±1 C_(t) value (cycle number where thefluorescence value crosses the threshold) when compared to all thesingleplex PCR's. Evaluation of the Spseudo and other P-LVS bacteriayielded results similar to the individual assays. There were noadditional positive or negative reactions.

Currently, the trend is to multiplex assays for simultaneous detectionof various pathogens, representing a savings in time and money. We havedone this for a single pathogen but would propose that our lytA-CDC orpsaA-CDC primer probe sets would provide high specificity in arespiratory platform multiplexed with primers and probes for detectionof other respiratory pathogens. This potential for multiplexing and thespeed of performance make these assays promising tools for moleculardetection and epidemiologic carriage studies. The use of this technologyshould offer an added advantage when used in conjunction with otherassays for pneumococcal disease diagnosis.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used, andit is intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds,chemical moieties, or examples described in conjunction with aparticular aspect, embodiment, or example of the invention are to beunderstood to be applicable to any other aspect, embodiment, or exampleof the invention. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims.

1. A method for detecting a Streptococcus pneumoniae nucleic acid in asample, comprising: contacting the sample with at least one probecomprising a nucleic acid sequence between 20 and 40 nucleotides inlength capable of hybridizing under very high stringency conditions to aStreptococcus pneumoniae nucleic acid sequence set forth as SEQ ID NO:13 or SEQ ID NO: 15, wherein the probe comprises a nucleic acid sequenceat least 95% identical to the nucleotide sequence set forth as SEQ IDNO: 5, SEQ ID NO: 6, or SEQ ID NO: 12; and detecting hybridizationbetween the Streptococcus pneumoniae nucleic acid and the probe, whereinthe detection of hybridization indicates the presence of theStreptococcus pneumoniae nucleic acid in the sample.
 2. The methodaccording to claim 1, wherein the probe consists essentially of thenucleic acid sequence set forth as SEQ ID NO: 5, SEQ ID NO: 6, or SEQ IDNO:
 12. 3. The method according to claim 2, wherein the probe consistsof X-GCCGAAAACGCTTGATACAGGGAG-Y (SEQ ID NO: 5), orX-TGCCGAAAACGCTTGATACAGGGAG-Y (SEQ ID NO: 6), wherein X is afluorophore, and wherein Y is a dark quencher or acceptor dye, orwherein the probe consists of X-CTCAAGT“T”GGAAACCACGAGTAAGAGTGATGAA-Y(SEQ ID NO: 12), wherein X is a fluorophore, Y is one or morephosphates, and “T” is a thymine with a dark quencher or acceptor dyelinked to it.
 4. (canceled)
 5. The method according to claim 1, whereinthe probe is labeled.
 6. The method according to claim 5, wherein theprobe is radiolabeled, fluorescently-labeled, biotin-labeled,enzymatically-labeled, or chemically-labeled.
 7. The method according toclaim 5, wherein the probe is labeled with a fluorophore, a fluorescencequencher, or a combination thereof.
 8. (canceled)
 9. The methodaccording to claim 5, wherein detecting hybridization comprisesdetecting a change in signal from the labeled probe during or afterhybridization relative to signal from the label before hybridization.10. The method according to claim 1, wherein the method discriminatesbetween Streptococcus pneumoniae nucleic acid and a pneumococcus-likeviridans Streptococci (P-LVS) nucleic acid.
 11. The method according toclaim 1, further comprising amplifying the Streptococcus pneumoniaenucleic acid by polymerase chain reaction (PCR), real-time PCR, reversetranscriptase-polymerase chain reaction (RT-PCR), real-time reversetranscriptase-polymerase chain reaction (rt RT-PCR), ligase chainreaction, or transcription-mediated amplification (TMA).
 12. (canceled)13. The method according to claim 11, wherein amplifying theStreptococcus pneumoniae nucleic acid comprises contacting the samplewith at least one primer between 15 and 40 nucleotides in length capableof hybridizing under very high stringency conditions to a Streptococcuspneumoniae nucleic acid sequence set forth as SEQ ID NO: 13 or SEQ IDNO: 15, wherein the primer is capable of amplifying the Streptococcuspneumoniae nucleic acid.
 14. The method according to claim 13, whereinthe primer comprises a nucleic acid sequence at least 95% identical tothe nucleotide sequence set forth as SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 10, or SEQ ID NO:
 11. 15. The method according to claim 14, whereinthe primer consists essentially of the nucleic acid sequence set forthas SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO:
 11. 16. Themethod according to claim 1, wherein the sample is a biological sampleobtained from a subject.
 17. The method of claim 16, wherein thepresence of a Streptococcus pneumoniae nucleic acid in the biologicalsample indicates the presence of a Streptococcus pneumoniae infection inthe biological sample obtained from the subject.
 18. (canceled)
 19. Themethod according to claim 1, wherein the probe is arrayed in apredetermined array with an addressable location.
 20. A probe for thedetection of a Streptococcus pneumoniae nucleic acid comprising anucleic acid sequence between 20 and 40 nucleotides in length capable ofhybridizing under very high stringency conditions to a Streptococcuspneumoniae nucleic acid sequence set forth as SEQ ID NO: 13 or SEQ IDNO: 15, wherein the probe comprises a nucleic acid sequence at least 95%identical to the nucleotide sequence set forth as SEQ ID SEQ ID NO: 5,SEQ ID NO: 6, or SEQ ID NO:
 12. 21. The probe according to claim 20,wherein the probe consists essentially of the nucleic acid sequence setforth as SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
 12. 22. The probeaccording to claim 21, wherein the probe consists ofX-GCCGAAAACGCTTGATACAGGGAG-Y (SEQ ID NO: 5), orX-TGCCGAAAACGCTTGATACAGGGAG-Y (SEQ ID NO: 6), wherein X is afluorophore, and wherein Y is a dark quencher or acceptor dye, orwherein the probe consists of X-CTCAAGT“T”GGAAACCACGAGTAAGAGTGATGAA-Y(SEQ ID NO: 12), wherein X is a fluorophore, Y is one or morephosphates, and “T” is a thymine with a dark quencher or acceptor dyelinked to it.
 23. (canceled)
 24. The probe according to claim 20,wherein the probe is labeled.
 25. The probe according to claim 24,wherein the probe is radiolabeled, fluorescently-labeled,biotin-labeled, enzymatically-labeled, or chemically-labeled.
 26. Theprobe according to claim 24, wherein the probe is labeled with afluorophore, a fluorescence quencher, or a combination thereof. 27.(canceled)
 28. A primer for the amplification of a Streptococcuspneumoniae nucleic acid sequence comprising a nucleic acid 15 to 40nucleotides in length capable of hybridizing under very high stringencyconditions to a Streptococcus pneumoniae nucleic acid sequence set forthas SEQ ID NO: 13 or SEQ ID NO: 15, comprising a nucleic acid sequence atleast 95% identical to a nucleic acid sequence set forth as SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, and wherein the primer iscapable of directing the amplification of the Streptococcus pneumoniaenucleic acid.
 29. The primer according to claim 28, wherein the primerconsists essentially of the nucleic acid sequence set forth as SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO:
 11. 30. A set of primersfor the amplification of a Streptococcus pneumoniae nucleic acidcomprising: one or more forward primers consisting essentially of thenucleic acid sequence set forth as SEQ ID NO: 3 or SEQ ID NO: 10; andone or more reverse primers consisting essentially of the nucleic acidsequence set forth as SEQ ID NO: 4 or SEQ ID NO: 11, wherein the set ofprimers is capable of hybridizing to and directing the amplification ofthe Streptococcus pneumoniae nucleic acid.
 31. (canceled)
 32. A kit fordetecting a Streptococcus pneumoniae nucleic acid in a sample,comprising: the probe according to claim 20; and instructions forhybridizing the probe to the Streptococcus pneumoniae nucleic acidwithin the sample.
 33. The kit according to claim 32, further comprisinga set of primers comprising one or more forward primers consistingessentially of the nucleic acid sequence set forth as SEQ ID NO: 3 orSEQ ID NO: 10; and one or more reverse primers consisting essentially ofthe nucleic acid sequence set forth as SEQ ID NO: 4 or SEQ ID NO: 11,wherein the set of primers is capable of hybridizing to and directingthe amplification of the Streptococcus pneumoniae nucleic acid.
 34. Adevice for detecting a Streptococcus pneumoniae nucleic acid in asample, comprising nucleic acid array comprising at least one probeaccording to claim
 20. 35. A method for diagnosing a Streptococcuspneumoniae infection in a subject suspected of having a Streptococcuspneumoniae infection comprising: obtaining a sample comprising nucleicacids from the subject; contacting the sample with one or more nucleicacid probes according to claim 20; detecting hybridization between aStreptococcus pneumoniae nucleic acid sequence present in the sample andthe probe, wherein the detection of hybridization indicates that thesubject is infected with Streptococcus pneumoniae.
 36. The methodaccording to claim 35, further comprising amplifying the Streptococcuspneumoniae nucleic acid with a set of primers comprising one or moreforward primers consisting essentially of the nucleic acid sequence setforth as SEQ ID NO: 3 or SEQ ID NO: 10; and one or more reverse primersconsisting essentially of the nucleic acid sequence set forth as SEQ IDNO: 4 or SEQ ID NO: 11, wherein the set of primers is capable ofhybridizing to and directing the amplification of the Streptococcuspneumoniae nucleic acid.